Genomic epidemiological analysis of Klebsiella pneumoniae from Portuguese hospitals reveals insights into circulating antimicrobial resistance.

Klebsiella pneumoniae (Kp) bacteria are an increasing threat to public health and represent one of the most concerning pathogens involved in life-threatening infections and antimicrobial resistance (AMR). To understand the epidemiology of AMR of Kp in Portugal, we analysed whole genome sequencing, susceptibility testing and other meta data on 509 isolates collected nationwide from 16 hospitals and environmental settings between years 1980 and 2019. Predominant sequence types (STs) included ST15 (n = 161, 32%), ST147 (n = 36, 7%), ST14 (n = 26, 5%) or ST13 (n = 26, 5%), while 31% of isolates belonged to STs with fewer than 10 isolates. AMR testing revealed widespread resistance to aminoglycosides, fluoroquinolones, cephalosporins and carbapenems. The most common carbapenemase gene was blaKPC-3. Whilst the distribution of AMR linked plasmids appears uncorrelated with ST, their frequency has changed over time. Before year 2010, the dominant plasmid group was associated with the extended spectrum beta-lactamase gene blaCTX-M-15, but this group appears to have been displaced by another carrying the blaKPC-3 gene. Co-carriage of blaCTX-M and blaKPC-3 was uncommon. Our results from the largest genomics study of Kp in Portugal highlight the active transmission of strains with AMR genes and provide a baseline set of variants for future resistance monitoring and epidemiological studies.

A phylogenomic approach for the analysis of colistin resistance-associated genes in Klebsiella pneumoniae, its mutational diversity and implications for phenotypic resistance.

The emergence of carbapenemase-producing Klebsiella pneumoniae strains has triggered the use of old antibiotics such as colistin. This is driving the emergence of colistin resistance in multidrug-resistant strains that underlie life-threatening infections. This study analyses the mutational diversity of 22 genes associated with colistin resistance in 140 K. pneumoniae clinical isolates integrated in a high-resolution phylogenetic scenario. Colistin susceptibility was accessed by broth microdilution. A total of 98 isolates were susceptible and 16 were resistant, 10 of which were carbapenemase producers. Across the 22 genes examined, 171 non-synonymous mutations and 9 mutations associated with promoter regions were found. Eighty-five isolates had a truncation and/or deletion in at least one of the 22 genes. However, only seven mutations, the complete deletion of mgrB or insertion sequence (IS)-mediated disruption, were exclusively observed in resistant isolates. Four of these (mgrBIle13fs, pmrBGly207Asp, phoQHis339Asp and ramAIle28Met) comprised novel mutations that are potentially involved in colistin resistance. One strain bore a ISEcp1-blaCTX-M-15::mgrB disruption, underlying co-resistance to third-generation cephalosporins and colistin. Moreover, the high-resolution phylogenetic context shows that most of the mutational diversity spans multiple phylogenetic clades, and most of the mutations previously associated with colistin resistance are clade-associated and present in susceptible isolates, showing no correlation with colistin resistance. In conclusion, the present study provides relevant data on the genetic background of genes involved with colistin resistance deeply rooted across monophyletic groups and provides a better understanding of the genes and mutations involved in colistin resistance.

First Description of Ceftazidime/Avibactam Resistance in a ST13 KPC-70-Producing Klebsiella pneumoniae Strain from Portugal.

The combination of ceftazidime/avibactam (CZA) is a novel ß-lactam/ß-lactamase inhibitor with activity against Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales. Emerging cases caused by CZA-resistant strains that produce variants of KPC genes have already been reported worldwide. However, to the best of our knowledge, no CZA-resistant strains were reported in Portugal. In September 2019, a K. pneumoniae CZA-resistant strain was collected from ascitic fluid at a surgery ward of a tertiary University Hospital Center in Lisboa, Portugal. The strain was resistant to ceftazidime/avibactam, as well as to ceftazidime, cefoxitin, gentamicin, amoxicillin/clavulanic acid, and ertapenem, being susceptible to imipenem and tigecycline. A hypermucoviscosity phenotype was confirmed by string test. Whole-genome sequencing (WGS) analysis revealed the presence of an ST13 KPC70-producing K. pneumoniae, a KPC-3 variant, differing in two amino-acid substitutions (D179Y and T263A). The D179Y mutation in the KPC Ω-loop region is the most common amino-acid substitution in KPC-2 and KPC-3, further leading to CZA resistance. The second mutation causes a KPC-70 variant in which threonine replaces alanine (T263A). The CZA-resistant strain showed the capsular locus KL3 and antigen locus O1v2. Other important virulence factors were identified: fimbrial adhesins type 1 and type 3, as well as the cluster of iron uptake systems aerobactin, enterobactin, salmochelin, and yersiniabactin included in integrative conjugative element 10 (ICEKp10) with the genotoxin colibactin cluster. Herein, we report the molecular characterization of the first hypervirulent CZA-resistant ST13 KPC-70-producing K. pneumoniae strain in Portugal. The emergence of CZA-resistant strains might pose a serious threat to public health and suggests an urgent need for enhanced clinical awareness and epidemiologic surveillance.

Whole-Genome Sequencing Enables Molecular Characterization of Non-Clonal Group 258 High-Risk Clones (ST13, ST17, ST147 and ST307) among Carbapenem-Resistant Klebsiella pneumoniae from a Tertiary University Hospital Centre in Portugal.

The carbapenem-resistant Enterobacterales (CRE) strains have been identified by the World Health Organization as critical priority pathogens in research and development of diagnostics, treatments, and vaccines. However, recent molecular information about carbapenem-resistant K. pneumoniae (CRK) epidemiology in Portugal is still scarce. Thus, this study aimed to provide the molecular epidemiology, resistome, and virulome of CRK clinical strains recovered from a tertiary care hospital centre (2019-2021) using polymerase chain reaction (PCR) and the advanced molecular technique whole-genome sequencing (WGS). PCR amplification of carbapenemase genes was performed in 437 carbapenem-resistant K. pneumoniae strains. The most frequent carbapenemases were: KPC-3 (42%), followed by OXA-181 (20%), GES-5 (0.2%), and NDM-1 (0.2%). Additionally, 10 strains (2%) coproduced KPC-3 and OXA-181, and 1 strain coproduced KPC-3 and OXA-48 (0.2%). The genomic population structure of 68 strains characterized by WGS demonstrated the ongoing dissemination of four main high-risk clones: ST13, ST17, ST147, and ST307, while no clones belonging to the European predominant clonal groups (CG15 and CG258) were found. Moreover, we describe one K. pneumoniae ST39-KL62 that coproduced the NDM-1 carbapenemase and the extended-spectrum beta-lactamase CTX-M-15, and one K. pneumoniae ST29-KL54 GES-5 and BEL-1 coproducer. Furthermore, a high prevalence of iron siderophores were present in all CRK strains, with several strains presenting both colibactin and the hypermucoviscosity phenotype. Thus, the data presented here highlight an uncommon molecular epidemiology pattern in Portugal when compared with most European countries, further supporting the emergence and dissemination of nonclonal group 258 hypervirulent multidrug high-risk clones and the need to promote in-depth hospital molecular surveillance studies.

Klebsiella pneumoniae and Colistin Susceptibility Testing: Performance Evaluation for Broth Microdilution, Agar Dilution and Minimum Inhibitory Concentration Test Strips and Impact of the "Skipped Well" Phenomenon.

The emergence of multidrug resistant Gram-negative pathogens, particularly carbapenemase producers, has forced clinicians to use last line antibiotics, such as colistin. Since colistin susceptibility testing presents several challenges, this study aimed at evaluating the performance of two alternative susceptibility methods for Klebsiella pneumoniae, namely, agar dilution (AD) and MIC test strips (MTS). These approaches were compared with the reference method, broth microdilution (BMD), and provide a quantitative description for the "skipped well" (SW) phenomenon. Colistin susceptibility was evaluated by BMD and AD in parallel and triplicate, using 141 K. pneumoniae clinical isolates while MTS performance was evaluated only for a subset (n = 121). Minimum inhibitory concentration analysis revealed that a substantial part (n = 26/141; 18.4%) of the initial isolates was deemed undetermined by BMD due to the following: discordance between replicates (1.4%); presence of multiple SWs (7.8%); and the combination of both events (9.2%). Both AD and MTS revealed a high number of false-susceptible strains ("very major errors"), 37.5% and 68.8%, respectively. However, AD agreement indices were reasonably high (EA = 71.3% and CA = 94.8%). For MTS these indices were lower, in particular EA (EA = 41.7% and CA = 89.6), but the approach enabled the detection of distinct sub-populations for four isolates. In conclusion, this study provides the most comprehensive study on the performance of AD and MTS for colistin susceptibility testing in K. pneumoniae, highlighting its limitations, and stressing the importance of sample size and composition. Further, this study highlights the impact of the SW phenomenon associated with the BMD method for K. pneumoniae.

Virulence and resistance determinants of Klebsiella pneumoniae isolated from a Portuguese tertiary university hospital centre over a 31-year period / Determinantes de virulencia y resistencia en Klebsiella pneumoniae aisladas en un centro hospitalario universitario en Portugal durante 31 años

INTRODUCTION: The rapid and complex evolution of bacterial resistance mechanisms in Klebsiella pneumoniae producing extended-spectrum β-lactamases and carbapenemases in Klebsiella pneumoniae is one of the most significant threats to public health. However, questions and controversies regarding the interactions between resistance and virulence in multidrug-resistant K. pneumoniae isolates remain unclear. METHODS: A retrospective cohort study was performed with 100 K. pneumoniae isolates recovered from a tertiary care university hospital centre in Lisbon over a 31-year period. Resistance and virulence determinants were screened using molecular methods (PCR, M13-PCR and MLST). RESULTS: The predominant virulence profile (fimH, mrkDv1, khe) was shared by all isolates, indicative of an important role of type 1 and 3 fimbrial adhesins and haemolysin, regardless of the type of β-lactamase produced. However, accumulation of virulence factors was identified in KPC-3-producers, with a higher frequency (p < 0.05) of capsular serotype K2 and iucC aerobactin when compared with non-KPC-3 Beta-lactamases or carbapenemases. Additionally, 9 different virulence profiles were found, indicating that the KPC-3 carbapenemase producers seem to adapt successfully to the host environment and maintain virulence via several pathways. CONCLUSION: This study describes an overlapping of multidrug-resistance and virulence determinants in ST-14K2 KPC-3 K. pneumoniae clinical isolates that may impose an additional challenge in the treatment of infections caused by this pathogen

INTRODUCCIÓN: La rápida y compleja evolución de los mecanismos de resistencia de Klebsiella pneumoniae productora de beta-lactamasas de espectro extendido y carbapenemasas en Klebsiella pneumoniae es una de las amenazas más importantes para la salud pública. Sin embargo, aun existe controversia sobre la interacción entre la resistencia y la virulencia en aislados de K. pneumoniae resistentes a múltiples antimicrobianos. MÉTODOS: Se realizó un estudio de cohorte retrospectivo con 100 aislados de Klebsiella pneumoniae de un centro hospitalario universitario en Lisboa durante 31 años. Los determinantes de la resistencia y virulencia se rastrearon utilizando métodos moleculares (PCR, M13-PCR y MLST). RESULTADOS: Todos los aislados compartían un perfil de virulencia predominante (fimH, mrkDv1, khe), lo que indica un papel importante de las adhesinas fimbriales de tipo 1 y 3, y de la hemolisina, independientemente del tipo de β-lactamasa producida. Sin embargo, la acumulación de factores de virulencia del serotipo capsular K2 y la aerobactina iucC se identificó con una mayor frecuencia en las cepas productoras de KPC-3 (p < 0,05) en comparación con las productoras de otras Beta-lactamasas o carbapenemasas. Además, se encontraron 9 perfiles de virulencia diferentes, indicativos de que las cepas productoras de carbapenemasa KPC-3 parecen adaptarse con éxito al entorno y mantener la virulencia por varias vías. CONCLUSIÓN: Este estudio describe la unión de resistencia a múltiples antimicrobianos junto con determinantes de virulencia en aislados clínicos de K. pneumoniae ST-14K2 KPC-3 lo que puede suponer un desafío adicional en el tratamiento de infecciones causadas por este patógeno

Community- and Hospital-Acquired Klebsiella pneumoniae Urinary Tract Infections in Portugal: Virulence and Antibiotic Resistance.

Klebsiella pneumoniae is a clinically relevant pathogen and a frequent cause of hospital-acquired (HA) and community-acquired (CA) urinary tract infections (UTI). The increased resistance of this pathogen is leading to limited therapeutic options. To investigate the epidemiology, virulence, and antibiotic resistance profile of K. pneumoniae in urinary tract infections, we conducted a multicenter retrospective study for a total of 81 isolates (50 CA-UTI and 31 HA-UTI) in Portugal. The detection and characterization of resistance and virulence determinants were performed by molecular methods (PCR, PCR-based replicon typing, and multilocus sequence typing (MLST)). Out of 50 CA-UTI isolates, six (12.0%) carried ß-lactamase enzymes, namely blaTEM-156 (n = 2), blaTEM-24 (n = 1), blaSHV-11 (n = 1), blaSHV-33 (n = 1), and blaCTX-M-15 (n = 1). All HA-UTI were extended-spectrum ß-lactamase (ESBL) producers and had a multidrug resistant profile as compared to the CA-UTI isolates, which were mainly resistant to ciprofloxacin, levofloxacin, tigecycline, and fosfomycin. In conclusion, in contrast to community-acquired isolates, there is an overlap between virulence and multidrug resistance for hospital-acquired UTI K. pneumoniae pathogens. The study is the first to report different virulence characteristics for hospital and community K. pneumoniae pathogens, despite the production of ß-lactamase and even with the presence of CTX-M-15 ESBL, a successful international ST15 clone, which were identified in both settings. This highlights that a focus on genomic surveillance should remain a priority in the hospital environment.

Virulence and resistance determinants of Klebsiella pneumoniae isolated from a Portuguese tertiary university hospital centre over a 31-year period.

INTRODUCTION: The rapid and complex evolution of bacterial resistance mechanisms in Klebsiella pneumoniae producing extended-spectrum ß-lactamases and carbapenemases in Klebsiella pneumoniae is one of the most significant threats to public health. However, questions and controversies regarding the interactions between resistance and virulence in multidrug-resistant K. pneumoniae isolates remain unclear. METHODS: A retrospective cohort study was performed with 100 K. pneumoniae isolates recovered from a tertiary care university hospital centre in Lisbon over a 31-year period. Resistance and virulence determinants were screened using molecular methods (PCR, M13-PCR and MLST). RESULTS: The predominant virulence profile (fimH, mrkDv1, khe) was shared by all isolates, indicative of an important role of type 1 and 3 fimbrial adhesins and haemolysin, regardless of the type of ß-lactamase produced. However, accumulation of virulence factors was identified in KPC-3-producers, with a higher frequency (p<0.05) of capsular serotype K2 and iucC aerobactin when compared with non-KPC-3 ß-lactamases or carbapenemases. Additionally, 9 different virulence profiles were found, indicating that the KPC-3 carbapenemase producers seem to adapt successfully to the host environment and maintain virulence via several pathways. CONCLUSION: This study describes an overlapping of multidrug-resistance and virulence determinants in ST-14K2 KPC-3 K. pneumoniae clinical isolates that may impose an additional challenge in the treatment of infections caused by this pathogen.

First Description of Colistin and Tigecycline-Resistant Acinetobacter baumannii Producing KPC-3 Carbapenemase in Portugal.

Herein, we describe a case report of carbapenem-resistant Acinetobacter baumannii and Klebsiella pneumoniae isolates that were identified from the same patient at a Tertiary University Hospital Centre in Portugal. Antimicrobial susceptibility and the molecular characterization of resistance and virulence determinants were performed. PCR screening identified the presence of the resistance genes blaKPC-3, blaTEM-1 and blaSHV-1 in both isolates. The KPC-3 K. pneumoniae isolate belonged to the ST-14 high risk clone and accumulated an uncommon resistance and virulence profile additional to a horizontal dissemination capacity. In conclusion, the molecular screening led to the first identification of the A. baumannii KPC-3 producer in Portugal with a full antimicrobial resistance profile including tigecycline and colistin.

[Syphilis: Prevalence in a Hospital in Lisbon]. / Sífilis: Prevalência num Hospital de Lisboa.

INTRODUCTION: Syphilis is a sexual and vertical transmitted disease. Its incidence is increasing in Europe, particularly, in Portugal. MATERIAL AND METHODS: A descriptive, retrospective study was performed based on positive treponemal tests from January to December 2013, at the Santa Maria Hospital, Lisbon. In-patients and out-patients evaluated in medical appointments and at the emergency department were included. We proceeded to epidemiological characterization, disease classification and definition of risk factors. RESULTS: We obtained a sample of 580 patients, of whom 51 with no clinical data and 45 with false positive serologies were excluded. There was a predominance of male patients (75%) and a mean age of 47 years. Most (59%) had syphilis successfully treated in the past and 3.7% were in follow-up. We recorded 13 primaries syphilis, 71 cases of secondary syphilis, 40 cases of early latent syphilis, 49 unknown duration syphilis and five cases of late latent syphilis. In the early syphilis group, 42% (n = 124) were HIV-positive and, in 8% both diagnosis were done simultaneously. DISCUSSION: We emphasize the high prevalence of syphilis/HIV co-infection in patients with early syphilis, reinforcing the importance of promoting the use of preventive measures. We obtained 11% of patients with late clinical forms, which are notifiable since June 2014, in Portugal. All serological tests for the diagnosis of syphilis have limitations which emphasizes the importance of clinical-laboratory correlation. CONCLUSION: Syphilis remains an important public health problem. It is necessary to establish education programs, screening and follow-up strategies to reduce their prevalence and to perform more efficient screening of the partners.

Introdução: A sífilis é uma doença de transmissão sexual e vertical. A sua incidência está a aumentar na Europa, particularmente em Portugal. Material e Métodos: Estudo retrospetivo baseado na análise laboratorial de testes treponémicos positivos, entre janeiro e dezembro de 2013, no Hospital de Santa Maria. Foram incluídos doentes internados, da consulta externa, do hospital dia e da urgência. Procedeu-se a caraterização epidemiológica, classificação da doença e de fatores de risco associados. Resultados: Obteve-se uma amostra de 484 doentes, após exclusão de 51 por ausência de dados clínicos nos processos e de 45 por valores falsos positivos. Verificou-se predomínio do sexo masculino (75%) e idade média de 47 anos. A maioria (59%) tinha testes serológicos compatíveis com sífilis no passado e 3,7% encontrava-se em vigilância clínica. Diagnosticou-se sífilis primária em 13doentes, secundária em 71, latente precoce em 40, latente indeterminada em 49 e latente tardia em cinco. No grupo sífilis recente, 42% (n = 124) eram seropositivos para o VIH e 8% tiveram, em simultâneo, este diagnóstico. Discussão: Salienta-se a elevada prevalência da coinfeção pelo VIH nos doentes com sífilis recente, reforçando a importância de promover a utilização de medidas preventivas. Registaram-se 11% de formas clínicas tardias, que são de notificação obrigatória desde junho de 2014. Todos os testes serológicos para o diagnóstico de sífilis apresentam limitações, o que enfatiza a importância da correlação clínico-laboratorial. Conclusão: A sífilis continua a ser um problema de saúde pública pelo que é necessário estabelecer programas de educação, rastreio e follow-up para reduzir a sua prevalência e tornar mais eficiente o rastreio dos parceiros.

Incidence of Burkholderia contaminans at a cystic fibrosis centre with an unusually high representation of Burkholderia cepacia during 15 years of epidemiological surveillance.

The Burkholderia cepacia complex (Bcc) is a heterogeneous group of bacteria comprising around 20 related species. These bacteria are important opportunistic pathogens, especially in cystic fibrosis (CF) patients, and are associated with a worse prognosis and decreased life expectancy. The taxonomic position of 20 Bcc isolates retrieved from CF patients receiving care at Hospital Santa Maria (HSM), in Lisbon, from 1995 to 2006, was re-examined in the present work. These isolates, formerly classified as Burkholderia cepacia (taxon K), are here reclassified as Burkholderia contaminans, including the former B. cepacia IST408, which was the focus of previous studies regarding the biosynthesis of the exopolysaccharide 'cepacian'. The CF population examined has been previously described as having an exceptionally high representation of B. cepacia, presumably due to a contamination arising from saline solutions for nasal application. Twenty-one additional isolates, obtained from a chronically infected patient, from 2006 to 2010, were also identified as B. contaminans. This study also provides insight into the potential clinical impact of B. contaminans, a species that is rarely associated with CF infections. Isolates belonging to this species were shown to be involved in chronic and transient respiratory infections, and were associated with severe lung function deterioration and with a case of death with cepacia syndrome. However, since the patients were co-infected with Burkholderia cenocepacia and other non-Burkholderia bacteria, the role played by B. contaminans is unclear. Nevertheless, B. contaminans isolates were found to prevail over B. cenocepacia isolates during co-infection of at least one chronically infected patient.

Burkholderia dolosa phenotypic variation during the decline in lung function of a cystic fibrosis patient during 5.5 years of chronic colonization.

Although rarely isolated from cystic fibrosis (CF) patients, Burkholderia dolosa is associated with accelerated lung function decline. During 18 years of epidemiological surveillance in the major Portuguese CF centre in Lisbon, only one patient was infected with B. dolosa. Pulmonary deterioration, associated with the evolution of forced expiratory volume in 1 s, occurred during 5.5 years of colonization with this B. dolosa clone (with the new sequence type ST-668). Transient co-colonization with Burkholderia cenocepacia and other bacterial and fungal pathogens occurred, but B. dolosa prevailed until the patient's death. The systematic assessment of relevant phenotypes for the sequential clonal isolates examined in this retrospective study (14 of B. dolosa and four of B. cenocepacia) showed that they were variants, although in general no isolation time-dependent pattern of alteration was identified. However, the first B. dolosa isolate retrieved was more susceptible to gentamicin, imipenem and tobramycin, and exhibited a higher swarming motility compared with most of the isolates obtained during the later stages of disease progression and antimicrobial therapy.

Extensive dissemination of methicillin-resistant Staphylococcus aureus (MRSA) between the hospital and the community in a country with a high prevalence of nosocomial MRSA.

According to the EARS-Net surveillance data, Portugal has the highest prevalence of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) in Europe, but the information on MRSA in the community is very scarce and the links between the hospital and community are not known. In this study we aimed to understand the events associated to the recent sharp increase in MRSA frequency in Portugal and to evaluate how this has shaped MRSA epidemiology in the community. With this purpose, 180 nosocomial MRSA isolates recovered from infection in two time periods and 14 MRSA isolates recovered from 89 samples of skin and soft tissue infections (SSTI) were analyzed by pulsed-field gel electrophoresis (PFGE), staphylococcal chromosome cassette mec (SCCmec) typing, spa typing and multilocus sequence typing (MLST). All isolates were also screened for the presence of Panton Valentine leukocidin (PVL) and arginine catabolic mobile element (ACME) by PCR. The results showed that ST22-IVh, accounting for 72% of the nosocomial isolates, was the major clone circulating in the hospital in 2010, having replaced two previous dominant clones in 1993, the Iberian (ST247-I) and Portuguese (ST239-III variant) clones. Moreover in 2010, three clones belonging to CC5 (ST105-II, ST125-IVc and ST5-IVc) accounted for 20% of the isolates and may represent the beginning of new waves of MRSA in this hospital. Interestingly, more than half of the MRSA isolates (8/14) causing SSTI in people attending healthcare centers in Portugal belonged to the most predominant clones found in the hospital, namely ST22-IVh (n = 4), ST5-IVc (n = 2) and ST105-II (n = 1). Other clones found included ST5-V (n = 6) and ST8-VI (n = 1). None of the MRSA isolates carried PVL and only five isolates (ST5-V-t179) carried ACME type II. The emergence and spread of EMRSA-15 may be associated to the observed increase in MRSA frequency in the hospital and the consequent spillover of MRSA into the community.

Survey of extended-spectrum beta-lactamases in Escherichia coli isolates from a Portuguese hospital and characterisation of a novel class 1 integron (In60A) carrying the blaCTX-M-9 gene.

Between November 2001 and November 2004, 231 Escherichia coli isolates resistant to beta-lactam antibiotics were identified. In 14 isolates, bla(TEM-24) (2 isolates), bla(TEM-52) (5 isolates) and bla(TEM-26) (7 isolates) were identified. In 145 E. coli isolates with the same M13 fingerprinting profile and the same resistance phenotype, the bla(CTX-M-15) gene was found in association with an insertion sequence ISEcp1. The bla(CTX-M-2) gene was identified in one E. coli isolate (290HSM), and in other E. coli isolate (246HSM) the bla(CTX-M-9) gene was contained in a new complex sul1-type class 1 integron (named In60A). This is the first report of three cefotaximases (CTX-M-15, CTX-M-2 and CTX-M-9) in E. coli isolates from a Portuguese hospital.

Direct application of the INNO-LiPA Rif.TB line-probe assay for rapid identification of Mycobacterium tuberculosis complex strains and detection of rifampin resistance in 360 smear-positive respiratory specimens from an area of high incidence of multidrug-resistant tuberculosis.

The INNO-LiPA Rif.TB assay for the identification of Mycobacterium tuberculosis complex strains and the detection of rifampin (RIF) resistance has been evaluated with 360 smear-positive respiratory specimens from an area of high incidence of multidrug-resistant tuberculosis (MDR-TB). The sensitivity when compared to conventional identification/culture methods was 82.2%, and the specificity was 66.7%; the sensitivity and specificity were 100.0% and 96.9%, respectively, for the detection of RIF resistance. This assay has the potential to provide rapid information that is essential for the effective management of MDR-TB.