A simple multiplex PCR for assessing prevalence of extended-spectrum β-lactamases producing Klebsiella pneumoniae in Intensive Care Units of a referral hospital in Shiraz,Iran

ObjectiveTo identify three common genes (bla_TEM, bla_SHV and bla_CTX-M) responsible for ESBL production in Klebsiella pneumoniae (K. pneumoniae)isolated from Intensive Care Units of Namazi Hospital, Shiraz, Iran.MethodsA total of 60 non-repetitive nosocomial isolates from 60 patients were selected during 2009-2010. The phenotypic identification of ESBL production was confirmed by Double Disk Synergy Test (DDST) according to CLSI guidelines. The ESBL's genotype was then analyzed by multiplex PCR of bla_TEM, bla_SHV and bla_CTX-M genes and DNA sequencing.ResultsThe primary susceptibility tests of K. pneumoniae showed that among 10 examined antibiotics, the most resistant and susceptible antibiotics identified in this study were ampicillin and imipenem, respectively. The phenotypic determination of ESBL by DDST showed that 60% (n=36) of isolates produced ESBL. Multiplex PCR of genes among K. pneumoniae isolates showed that 39% (n=18) of them have TEM, 39% (n=18) of them have both CTX-M and TEM and 13% (n=8) of them have TEM, SHV, CTX-M.ConclusionsOur findings reveal the high prevalence (60%) of ESBL producing K. pneumoniae from ICU patients along with a new pattern of bla_TEMdistribution differ from other countries.     

Diversity of genotypes in CTX-M-producing Klebsiella pneumoniae isolated in different hospitals in Brazil

ObjectiveThe present study was undertaken to characterize CTX-M ESBL-producing Klebsiella pneumoniae collected from hospitals in different cities of Brazil.Material and MethodsEighty-five K. pneumoniae strains isolated from hospitalized patients in six different hospitals of three cities of Brazil were analyzed. ESBL production was confirmed by the standard double-disk synergy test and the Etest^®. The MIC₅₀ and MIC₉₀ for ESBL-producing isolates were determined by the Etest^® method. The antimicrobial susceptibilities of bacterial isolates were determined using the agar diffusion method according to the CLSI. Screening for bla_TEM, bla_SHV, bla_CTX-M genes and class 1 integron was performed by PCR amplification. To determine the genomic diversity of CTX-M-producers, isolates were analyzed by macrorestriction profile analysis following PFGE.Results and DiscussionSeventy-one K. pneumoniae isolates were ESBL-producing. PCR and sequencing experiments detected 38 CTX-M-producing K. pneumoniae belonged to groups CTX-M 1, CTX-M 2, CTX-M 8 and CTX-M 9. The association of different types ESBL (CTX-M, SHV and TEM) was frequent. All K. pneumoniae isolates carried class 1 integron. PFGE analysis revealed thirty-one clonal types among CTX-M-producing isolates. The data presented herein illustrate the diversity of genotypes of CTX-M producing K. pneumoniae among Brazilians hospitals.     

Molecular epidemiology of CTX-M producing Enterobacteriaceae isolated from bloodstream infections in Rio de Janeiro,Brazil: emergence of CTX-M-15

ObjectiveThe present study was designed to evaluate the molecular epidemiology of CTX-M producing Klebsiella pneumoniae, Enterobacter cloacae and Escherichia coli isolated from bloodstream infections at tertiary care hospitals in the State of Rio de Janeiro, Brazil.Material and methodsA total of 231 nonduplicate Enterobacteriaceae were isolated from five Brazilian hospitals between September 2007 and September 2008. The antimicrobial susceptibility testing was performed by disk diffusion method according to the Clinical Laboratory Standard Institute. Isolates showing resistance to third-generation cephalosporins were screened for ESBL activity by the double-disk synergy test. The presence of bla_CTX-M, bla_CTX-M-15 and bla_KPC genes was determined by Polymerase Chain Reaction (PCR) amplification and DNA sequencing. The molecular typing of CTX-M producing isolates was performed by pulsed-field gel electrophoresis (PFGE).Results and discussionNinety-three isolates were screened as ESBL positive and 85 (91%) were found to carry CTX-M-type, as follows: K. pneumoniae 59 (49%), E. cloacae 15 (42%), and E. coli 11 (15%). Ten isolates resistant for carbapenems in K. pneumoniae were bla_KPC-2 gene positive. Among CTX-M type isolates, CTX-M-15 was predominant in more than 50% of isolates for K. pneumoniae, E. coli, and E. cloacae. PFGE analysis of CTX-M producing isolates showed the predominance of CTX-M-15 in 10 of 24 pulsotypes in K. pneumoniae, 6 of 13 in E. cloacae and 3 of 6 in E. coli. CTX-M-15 was also predominant among KPC producing isolates. In conclusion, this study showed that CTX-M-15 was circulating in Rio de Janeiro state in 2007–2008. This data reinforce the need for continuing surveillance because this scenario may have changed over the years.     

Characterization of cephalosporin-resistant clinical Enterobacteriaceae for CTX-M ESBLs in Bahrain

ObjectiveTo detect the presence of specific CTX-M class of extended spectyum β-lactamases (ESBLs) in a collection of cephalosporin-resistant Enterobacteriaceae isolates from Bahrain.MethodsA subset of 80 cephalosporin-resistant Enterobacteriaceae collected from Salmaniya Medical Complex, Bahrain, were characterized further for the presence of specific genogroups of CTX-M β-lactamases by multiplex- and monoplex- PCRs. The primers used for the multiplex and monoplex PCRs were of genogroups- 1, 2, 8, 9 and 25. Sequencing of the representative isolates was performed to find the circulating CTX-M-types.ResultsA total of 93.8% (75/80) isolates showed the amplicons corresponding to any of the genogroups (1, 2, 8, 9, 25) and the remaining 6.2% isolates turned out negative in multiplex PCR. Some of the isolates demonstrated multiple bands corresponding to the sizes of different genogroups. Further confirmation with respective monoplex PCR on these 75 isolates demonstrated that 93.3% (70/75) harbored CTX-M genogroup-1 and 6.7% (5/75) harbored genogroup-9. We did not find the presence of genogroups 2, 8, and 25 in these isolates by monoplex PCR. Sequencing results of genogroup-1 isolates demonstrated the presence of CTX-M-15-like ESBL, however, discrepant results were noticed in genogroup-9 isolates, sequencing showed them as CTX-M-55-like ESBL.ConclusionsThis is the first report from Bahrain characterizing the CTX-M genogroups of ESBLs and reporting the emergence of bla_CTX-M-55-like gene in this region.     

bla(CTX-M), bla(TEM), and bla(SHV) in Enterobacteriaceae from North-Indian tertiary hospital: high occurrence of combination genes

ObjectiveTo delineate the frequency of occurrence of bla_CTX-M, bla_TEM, and bla_SHV in Enterobacteriaceae from North-Indian tertiary hospital.MethodsA random collection of a subset of 45 Escherichia coli (E. coli) and 28 Klebsiella pneumoniae (K. pneumoniae) that was resistant to a third generation cephalosporin and obtained during 2007–2008 was selected for detailed screening for bla_CTX-M, bla_TEM, and bla_SHV by monoplex PCRs. The isolates demonstrating the presence of bla_CTX-M alleles were characterized for the specific CTX-M-genogroup by using a multiplex PCR.ResultsResistance to cefoperazone, ceftazidime, ceftriaxone, cefotaxime, cefoxitin and piperacillin was 100% each in K. pneumoniae isolates, whereas these resistance-rates for E. coli isolates were 93.1%, 83.8%, 91.9%, 93.6%, 97.3% and 97.1%, respectively. Concomitant resistance to aminoglycosides, quinolones and aztreonam was also noticed. Presence of any of the bla genes (bla_CTX-M, bla_TEM, and bla_SHV) was noticed in a total of 28 (38.4%) isolates of the 73 isolates studied. Many isolates demonstrated occurrence of these genes in various combinations. bla_CTX-M, bla_TEM, and bla_SHV were noticed in 28.8%, 10.9% and 13.7% isolates, respectively. Multiplex PCR in bla_CTX-M harboring isolates demonstrated the presence of CTX-M-Genogroup-1 and sequencing for the specific CTX-M-type revealed presence of CTX-M-15 type. RAPD typing showed wide diversity in isolates.ConclusionsThis is amongst the premier report describing the simultaneous occurrence of bla_TEM, bla_SHV, and bla_ampC in Indian Enterobacteriaceae and that wider dissemination of these genes, as demonstrated by diversity of isolates, raises concern and emphasizes a need for extensive search for the presence of these gene pools in Indian subcontinent.     

Nosocomial and community infections due to class A extended-spectrum β-lactamase (ESBLA)-producing Escherichia coli and Klebsiella spp. in southern Brazil

ObjectivesTo determine the prevalence of class A extended spectrum β-lactamases (ESBL)-producing Escherichia coli and Klebsiella spp., and to investigate clonality among ESBL-producing isolates of nosocomial and community infections. Methods: The study involved 354 nosocomial infections samples and 992 community infections samples, obtained between 2003 and 2006 at Caxias do Sul, RS. The detection of ESBL was performed by the disk-diffusion test. Presence of bla_CTX-M, bla_SHV and bla_TEM β-lactamase genes was evaluated by PCR, and genomic typing was determined by pulsed-field gel electrophoresis analysis. Results: Higher frequency of ESBL-producing isolates were detected among nosocomial samples of E. coli (6.7%) and Klebsiella (43.7%), than those obtained from community infections (0.4% and 2.6%). bla_TEM and bla_CTX were the most prevalent ESBL gene families in both E. coli and Klebsiella isolates. Different pulsotypes were obtained among ESBL-producing E. coli and 11 clones for Klebsiella spp., which occurred over the years and in different hospital wards. Among ESBL-producing K. pneumoniae, 74.3% transferred ESBL genes by conjugation and exhibited concomitant decreased aminoglycosides susceptibility. Conclusion: ESBL-producing E. coli, and especially K. pneumoniae are essentially a nosocomial problem, and their dissemination to the community is relatively limited. The great genetic variability observed among ESBL-producing bacteria indicates polyclonal spread and high transference of ESBL genes between bacteria in the hospital environment. This information is of paramount importance for nosocomial infection control.     

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

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

Activity of ceftolozane-tazobactam and comparators against gram-negative bacilli: Results from the study for monitoring antimicrobial resistance trends (SMART – Brazil; 2016–2017)

Multi-drug resistant Gram-negative bacilli (GNB) have been reported as cause of serious hospital-acquired infections worldwide. The aim of this study was to investigate the in vitro activity of ceftolozane-tazobactam compared to other agents against GNB isolated from patients admitted to Brazilian medical centers between the years 2016 and 2017. Presence of β-lactamase encoding genes was also evaluated.MethodsAntimicrobial susceptibility testing of GNB isolated from intra-abdominal (IAI), respiratory (RTI), and urinary tract infections (UTI) was performed according to ISO 227-1 guidelines and interpreted following CLSI and BrCAST/EUCAST guidelines. Qualifying Enterobacteriaceae isolates were screened for the presence of β-lactamase genes by PCR followed by DNA sequencing.Results1748 GNB collected from UTI (45.2%), IAI (25.7%) and RTI (29.1%) were evaluated. Ceftolozane-tazobactam remained highly active (94.7%) against E. coli isolates. Among K. pneumoniae, susceptibility rates were 85.9% and 85.4% for amikacin and colistin, whereas ceftolozane-tazobactam (44.1% susceptible) and carbapenems (55.2-62.2% susceptible) showed poor activity due to bla_KPC-2. Against E. cloacae amikacin, imipenem, and meropenem retained good activity (>90%). Ceftolozane-tazobactam was the most potent β-lactam agent tested against P. aeruginosa (90.9% susceptible), including ceftazidime and imipenem resistant isolates. β-lactamase encoding genes testing was carried out in 433 isolates. bla_CTX-M variants were predominant in E. coli, P. mirabilis and E. cloacae. Among the K. pneumoniae molecularly tested, most carried bla_KPC (68.5%), with all harboring bla_KPC-2, except two isolates carrying bla_KPC-3 or bla_KPC-30. ESBL encoding genes, mainly CTX-M family, were frequently detected in K. pneumoniae, plasmid-mediated AmpC were rare. A variety of PDC encoding genes were detected in P. aeruginosa isolates with five isolates harboring MBL and one KPC encoding genes.ConclusionCeftolozane-tazobactam was very active against E. coli, P. mirabilis and P. aeruginosa isolates and could constitute an excellent therapeutic option including for those isolates resistant to extended-spectrum cephalosporins and carbapenems but not producers of carbapenemases.     

Molecular characterization and in vitro susceptibilities of β-lactamase producing Escherichia coli,Klebsiella species,Acinetobacter baumannii,Pseudomonas aeruginosa and Staphylococcus aureus to CSE1034 and other β-lactams

ObjectiveTo study the prevalence of extended-spectrum β-lactamases (ESBLs) among 663 clinical isolates obtained from various parts of India and to study the occurrence of different variants of ESBLs among these isolates.MethodsPhenotypic characterization and susceptibility studies were performed according to the methods described in Clinical and Laboratory Standards Institute guidelines. The occurrence of ESBL variants was analyzed with PCR using the previously reported primers.ResultsAmong the six hundred sixty three isolates, the identified isolates were Acinetobacter baumannii (72), Escherichia coli (218), Klebsiella pneumoniae (30), Klebsiella oxytoca (63), Pseudomonas aeruginosa (264) and Staphylococcus aureus (16). PCR results revealed that approximately 89.0% of Pseudomonas aeruginosa isolates were positive for ESBL followed by Escherichia coli (85.3%), Klebsiella pneumoniae (76.6%), Klebsiella oxytoca (73.0%), Acinetobacter baumannii (72.2%) and Staphylococcus aureus (31.2%). The overall prevalence of ESBL was 82.5%. The presence of TEM type ESBLs were the predominant (in 186 isolates), followed by SHV (138), OXA (92), CTX-M (65), AmpC (33), KPC (28) and blaZ (5). Of the drugs involved in the study, CSE1034 was found to be the most efficacious against all of ESBL positive clinical isolates showing susceptibility approximately 95.7% with minimal inhibitory concentration values between 0.125 and 8.000 μg/mL for all strains tested. The susceptibilities of penems (meropenem and imipenem and cilastatin) ranged between 83% and 93% for all the isolates. The susceptibilities of other drugs like piperacillin and tazobactam, amoxicillin and clavulanic acid, cefoperazone and sulbactam were <45% for all the isolates.ConclusionsResults of the present study indicated that majority of the isolates was susceptible to CSE1034 and it could be a potent antibacterial agent for the treatment of severe bacterial infections caused by such organisms.     

High prevalence of CTX-M-1 group in ESBL-producing enterobacteriaceae infection in intensive care units in southern Chile

Enterobacteria-producing extended-spectrum β-lactamases (ESBL) play an important role in healthcare infections, increasing hospitalization time, morbidity and mortality rates. Among several ESBLs that emerge from these pathogens, CTX-M-type enzymes had the most successful global spread in different epidemiological settings. Latin America presents high prevalence of CTX-M-2 in ESBL-producing enterobacterial infections with local emergence of the CTX-M-1 group. However, this high prevalence of the CTX-M-1 group has not yet been reported in Chile. The aim of this study was to identify ESBLs among enterobacteria isolated from clinical samples of critically ill patients from southern Chile. One-hundred thirty seven ESBL-producing bacteria were isolated from outpatients from all critical patient units from Hernán Henríquez Aravena Hospital. Phenotype characterization was performed by antibiogram, screening of ESBL, and determination of minimum inhibitory concentration (MIC). PCR was used for genetic confirmation of resistance. Molecular typing was performed by ERIC-PCR. ESBL-producing isolates were identified as Klebsiella pneumoniae (n = 115), Escherichia coli (n = 18), Proteus mirabilis (n = 3), and Enterobacter cloacae (n = 1), presenting multidrug resistance profiles. PCR amplification showed that the strains were positive for blaSHV (n = 111/81%), blaCTX-M-1 (n = 116/84.7%), blaTEM (n = 100/73%), blaCTX-M-2 (n = 28/20.4%), blaCTX-M-9 (0.7%), blaPER-1 (0.7%), and blaGES-10 (0.7%). The multiple production of ESBL was observed in 93% of isolates, suggesting high genetic mobility independent of the clonal relationship. The high frequency of the CTX-M-1 group and a high rate of ESBL co-production are changing the epidemiology of the ESBL profile in Chilean intensive care units. This epidemiology is a constant and increasing challenge, not only in Chile, but worldwide.     

Prevalence of plasmid mediated bla(TEM-1) and bla(CTX-M-15) type extended spectrum beta-lactamases in patients with sepsis

ObjectiveTo characterize the bacterial pathogens in patients having gram negative septicaemia. Further, to evaluate the antimicrobial resistance and underlying molecular mechanisms in these strains.MethodsA total number of 70 cases of gram negative sepsis were included in this prospective, open labeled, observational study. Standard methods for isolation and identification of bacteria were used. Antimicrobial susceptibility and ESBL testing was performed by the standard disc diffusion method. PCR amplification was performed to identify bla_CTX-M, bla_SHV and bla_TEM type ESBLs. Conjugation experiments were performed to show resistant marker transfer.ResultsThe most prevalent isolates Escherichia coli (E. coli) 58.6%, Klebsiella Spp. 32.9% and Pseudomonas 8.6%, were resistant to most of the antimicrobials including cefazolin, ceftriaxone, cefuroxime, ampicillin and co-trimoxazole but sensitive to imipenem and meropenem. ESBL and MBL production was seen 7.3% and 12.2% of E. coli isolates respectively. Three isoaltes were found to have bla_CTX-M-15 and two of them also showed bla_TEM-1 type enxyme. Whereas, none of them showed bla_SHV. Conjugation experiments using J-53 cells confirmed these resistant markers as plasmid mediated.ConclusionsThis work highlights the molecular epidemiology of escalating antimicrobial resistance and likely switch over of bla_CTX-M-15 type extended spectrum beta-lactamases by bla_TEM type ESBLs in India. Further, the antimicrobial resistance by horizontal gene transfer was predominant among Enterobacteraceae in the community setting.     

Prevalence of extended-spectrum beta-lactamase and class 1 integron integrase gene intl1 in Escherichia coli from Thai patients and healthy adults

Among 120 Escherichia coli isolates from Thai patients, 37 and 9 isolates were extended-spectrum beta-lactamase (ESBL) and suspected ESBL producers respectively while 5 E. coli isolates from 120 Thai healthy adults were suspected ESBL producers. Integrase (intl1) gene was detected in 99% of the clinical and 87% of the non-clinical isolates. Among 37 ESBL producers, percent recovery of bla(TEM), bla(CTX-M), bla(SHV) and bla(VEB) was 78%, 78%, 8% and 8%, respectively. Twenty-five isolates of ESBL producers carried both bla(TEM) and bla(CTX-M), 2 isolates carried 3 genes (bla(TEM), blac(CTX-M), and bla(SHV)) and 3 showed no detectable bla gene. Among the 14 suspected ESBL producers, intl1 and bla(TEM) were detected in 13 isolates. ESBL producers from clinical samples were resistant to most of the tested antimicrobial agents compared to non-ESBL producers and isolates from healthy adults with about half of the latter susceptible to all tested antimicrobial agents. Only one clinical isolate was resistant to imipenem. Susceptibility to trimethoprim/sulfamethoxazole among the clinical isolates in ESBL producer group (27%) and non-producer group (33%) were comparable, whereas the percent susceptibility of the non-clinical isolates was about twice that of the clinical isolates.     

Comparative study on occurrence of class A and class C β -lactamase genes and their co-occurrence in Indian Enterobacteriaceae during years 2009 and 2010

ObjectiveTo determine the occurrence of class A and class C β-lactamase genes and their co-occurrence in Indian Enterobacteriaceae.Methods52 third generation cephalosporin resistant isolates were phenotypically detected by combination disk method and screened by PCR to identify class A and class C type β-lactamase genes.ResultsOf the 52 isolates, 94.2% (49) were found harboring any of the bla_ESBLs. bla_CTX-M, bla_SHV and bla_TEM were present in 82.6% (43/52), 59.6% (31/52), and 42.3% (22/52) isolates, respectively. Of the 49 ESBL positive isolates 57.1% (28/49) showed co-occurrence of bla_ampC with bla_ESBLs. On the contrary, the collection from 2009 showed their co-occurrence in 81.4% isolates.ConclusionsThe comparative study shows a downward trend for co-existence of bla_ESBLs with bla_ampC from 2009 to 2010. Further large scale studies are needed to address the co-occurrence of class A and class C β-lactamases in India and the resistance trend occurring over a period of time.     

Colonization of intestinal microbiota with carbapenemase-producing Enterobacteriaceae in paediatric intensive care units in Cairo,Egypt

Background and study aimsColonized patients with carbapenamase producing Enterobacteriaceae (CPE) are vulnerable to invasive infections from their endogenous flora. We aimed to assess faecal colonization with (CPE) among children admitted to Cairo University paediatric intensive care units (ICUs). The phenotypic and genotypic characterizations of carbapenemase-producing Enterobacteriaceae were also studied.Patients and methodsA total of 413 Enterobacteriaceae isolates have been isolated from cultured rectal swabs of 100 children. All swabs were inoculated on ChromID™ CARBA agar to screen for carbapenem resistant Enterobacteriaceae (CRE). Disk diffusion method, Modified Hodge test (MHT) and further genotypic detection of carbapenemases genes (bla_OXA-48, bla_KPC and bla_NDM-1, bla_VIM and bla_IMP) by multiplex PCR were done.ResultsOut of 413 Enterobacteriaceae isolates; 100 isolates were defined as CRE. Bla_OXA-48 was detected in (33%); Escherichia coli (n = 11), Klebsiella oxytoca (n = 3) and Klebsiella pneumoniae (n = 19), while (27%) carried bla_NDM-1 Escherichia coli (n = 7), and Klebsiella pneumoniae (n = 20).ConclusionPrevalence of carbapenem resistant Enterobacteriaceae was 24%, various genes of carbapenemases were detected in 80% of carbapenem resistant Enterobacteriaceae with dominance of bla_OXA-48. Understanding the colonization status of our patients with strict infection control measures can reduce the risk of horizontal gene transfer of carbapenemases.     

Increasing secondary bacterial infections with Enterobacteriaceae harboring bla(CTX-M-15) and bla(CMY-6) in patients with bronchogenic carcinoma: an emerging point of concern

ObjectiveTo look for secondary bacterial infections in bronchogenic carcinoma (BC_A) with resistant organisms harboring bla genes considering the paucity of relevant studies.MethodsA total of 137 confirmed cases of BC_A and 34 healthy volunteers were studied for the occurrence and prevalence of bla_CTX-M and and bla_AmpC harboring–enterobacteriaceae. A subset of these patients (n=69) was previously reported for the secondary infection with the Aspergillus species. Bronchoalveolar lavages (BAL) were subjected for bacterial and fungal cultures and the bacterial isolates were screened by multiplex PCRs for the presence of bla_CTX–M and bla_AmpC. The isolates were also screened for the association of insertion sequence (IS26) by PCR and characterized by RAPD for any clonal relatedness.ResultsA total of 143 bacterial isolates were obtained from 137 BAL specimens of BC_A patients. The Enterobacteriaceae–isolates were multidrug-resistant showing concomitant resistance to fluoroquinolones and aminoglycosides. Both bla_CTX-M and bla_AmpC of CIT family were detected in 77.4% and 27.4% isolates, respectively. Sequencing revealed the presence of bla_CTX–M–15 and bla_CMY-6. Twenty one percent of the isolates were simultaneously harboring bla_ampC and bla_CTX–M–15. IS26 PCR and RAPD typing revealed the presence of diverse bacterial population but no predominant clone was identified. The present study also suggests strong association of aspergillosis with lung cancer and further strengthens the potential use of non-validated serological tests suggested earlier.ConclusionsWe emphasize that all patients of bronchogenic carcinoma should also be screened for secondary bacterial infections, along with secondary fungal infections, so as to introduce early and specific antimicrobial therapy and to prevent unwanted deaths.     

An observational study on bloodstream extended-spectrum beta-lactamase infection in critical care unit: incidence, risk factors and its impact on outcome

BackgroundThe incidence of nosocomial infections caused by extended-spectrum beta-lactamase (ESBL) producing microbes is increasing rapidly in the last few years. However, the clinical significance of infections caused by ESBL-producing bacteria in ICU patients remains unclear. We did a prospective study to look for incidence, risk factors and outcome of these infections in ICU patients.MethodsConsecutive isolates of Escherichia coli and Klebsiella pneumoniae in blood cultures were included for the analysis. Patients were divided into two groups based on the production of ESBL. Primary outcome measure was ICU mortality. Logistic regression analysis was done to identify risk factors for ESBL production.ResultsAmong the 95 isolates tested, 73 (76.8%) produced ESBL. Transfer from other hospitals or wards (OR 3.65; 95% CI: 1.3–10.1 and RR 1.35; 95% CI: 1.05–1.73) and previous history of antibiotics usage (OR 3.54; 95% CI: 1.04–11.97 and RR 1.5; 95% CI: 0.89–2.5) were risk factors for ESBL production. There was no significant difference in ICU mortality (p = 0.588), need for organ support between two groups.ConclusionThere is a high incidence of ESBL producing organisms causing blood stream infections in critically ill patients. Transfer from other hospitals and previous antibiotic usage are important risk factors for ESBL production. However ESBL production may not be associated with a poorer outcome if appropriate early antibiotic therapy is instituted.     

Variabilidad genética de Klebsiella pneumoniae con carbapenemasa tipo KPC proveniente de diferentes estados de Venezuela

Introduction

In Venezuela, there have been some reports of carbapenemase KPC-producing Klebsiella pneumoniae. Nevertheless, since the first report in 2008, only a few studies have been done on their molecular epidemiology in this country.

Faecal carriage of extended‐spectrum β‐lactamase‐producing Enterobacteriaceae among humans in Java,Indonesia, in 2001–2002

Objective To characterise commensal Escherichia coli and other Enterobacteriaceae with reduced susceptibility to cefotaxime that were collected in a large survey carried out among 3995 patients and healthy persons in two urban regions on Java, Indonesia, in 2001–2002. Methods The putative extended‐spectrum β‐lactamase (ESBL)‐producing Enterobacteriaceae were analysed using double‐disk synergy tests, isoelectric focusing, PCR assays, DNA sequencing, and pulsed‐field gel electrophoresis (PFGE). Results On the day of discharge after five or more days of hospitalisation, at least 95 of 999 (9.5%) patients carried ESBL‐positive Enterobacteriaceae as dominant faecal flora. Six patients were simultaneously colonised with E. coli and Klebsiella pneumoniae isolates with ESBL activity. On admission, only 6 of 998 (0.6%) patients were colonised. Faecal carriage of ESBL‐producing Enterobacteriaceae among healthy persons or persons visiting a public health centre was not detected. The 107 ESBL‐positive strains included 68 E. coli, 35 K. pneumoniae, and four other Enterobacteriaceae. bla_CTX‐M‐15 was the most prevalent ESBL in both E. coli (47.1%) and K. pneumoniae (45.7%), but the E. coli O25b‐ST131 clone was virtually absent. Other ESBL types found were: SHV‐2, ‐2a, ‐5, ‐12, CTX‐M‐3, ‐9, ‐14, and TEM‐19. PFGE revealed extensive genetic diversity among the isolates. Conclusions In 2001–2002, faecal carriage of ESBL‐producing Enterobacteriaceae as dominant flora in Indonesia was almost exclusively hospital‐associated. The presence of various bla_ESBL genes and the extensive genetic diversity among isolates argue against a single/dominant strain outbreak.     

Detection and molecular genetics of extended-spectrum beta-lactamases among cefuroxime-resistant Escherichia coli and Klebsiella spp. isolates from Finland, 2002-2004

Extended-spectrum beta-lactamase (ESBL) producing Escherichia coli and Klebsiella spp. isolates are spreading and becoming an increasing problem concerning treatment, diagnostics and hospital hygiene. We wanted to discover which genotypes are occurring in Finland and to assess the CLSI screening method. The isolates were collected from 26 laboratories during a 3-y period from 2002 to 2004. We studied the zone diameters by disk diffusion according to CLSI recommendations. ESBL genes were detected by PCR and the TEM and SHV genes were sequenced traditionally, while the CTX-M isolates were analysed with pyrosequencing. Of the 402 isolates included in the study, 269 (67%) were confirmed to be ESBL producers according to the CLSI criteria. The CTX-M genes were the most prevalent, especially the combination of a CTX-M-1-group and a TEM-1 gene. In our material there were few isolates that had an ESBL gene but were negative in the CLSI ESBL confirmatory test. During recent y especially the CTX-M producing isolates have increased in Europe and now they are also found in Finland with increasing prevalence.     

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

IntroductionThe 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.MethodsA 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).ResultsThe predominant virulence profile (fimH, mrkD_v1, 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 β-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.ConclusionThis study describes an overlapping of multidrug-resistance and virulence determinants in ST-14^K2 KPC-3 K. pneumoniae clinical isolates that may impose an additional challenge in the treatment of infections caused by this pathogen.