Bla SHV Research Articles

Detección de genes BLA en aislados clínicos de Escherichia coli provenientes de pacientes del Hospital Nacional Zacamil de El Salvador

La producción de enzimas inactivantes tipo Betalactamasas es uno de los mecanismos más importantes de resistencia antimicrobiana reportado a nivel mundial. Las infecciones ocasionadas por microorganismos productores de Betalactamasas de espectro extendido se relacionan con mayor mortalidad. Debido a la capacidad de diseminarse a través de elementos transponibles, los genes bla se reportan ampliamente. Sin embargo, existen países sin reportes de la circulación de éstos, a pesar de su importancia clínica.Este estudio provee las primeras evidencias de infecciones por Gram negativos intrahospitalarios asociados a genes de betalactamasas en El Salvador. Objetivo: Determinar la presencia de genes bla tipo TEM, SHV, CTX-M1-2 y AmpC en aislados clínicos de Escherichia coli provenientes de pacientes del Hospital Nacional Zacamil. Método: Se estudiaron 30 aislados consecutivos de Escherichia coli provenientes de pacientes durante el periodo de enero a mayo del 2015. La confirmación de especie y determinación de resistencia, se realizó con PromptTM Inoculation Sistem-D para equipo MicroScan®. La producción de BLEE se determinó por método de sinergia de doble disco. Se verificó la presencia de genes bla por medio de Reacción en Cadena de las Polimerasas. Resultados: 24 muestras resultaron positivas a BLEE determinado por método de Doble Disco y 20 por método automatizado MicroScan®. Siendo más efcaz el método de doble disco para la determinación fenotípica de producción de BLEE. Ciprofloxacina y cefriazona fueron los antibióticos más utilizados como tratamiento. Conclusión: El 36.6% de aislados resultó positivo a genes bla tipo TEM. Se obtuvo resultados negativos a los otros genes estudiados.CREA CIENCIA Vol. 10 No 2 ISSN1818-202X julio-diciembre 2016, p. 6-18

First study on characterization of virulence and antibiotic resistance genes in verotoxigenic and enterotoxigenic E. coli isolated from raw milk and unpasteurized traditional cheeses in Romania.

The study focused on the incidence of enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC) in raw milk and traditional dairy cheeses marketed in Romania, characterizing the virulence and antibiotic resistance genes of these isolates. One hundred and twenty samples of raw milk and 80 samples of unpasteurized telemy cheese were collected and cultured according to the international standard protocol. All the characteristic E. coli cultures were analyzed for the presence of STa, STb, LT, stx1, and stx2 toxicity genes. The ETEC/VTEC strains were tested for the presence of antibiotic resistance genes, such as aadA1, tetA, tetB, tetC, tetG, dfrA1, qnrA, aaC, sul1, bla SHV , bla CMY , bla TEM , and ere(A), using PCR. The results showed that 27 samples (18.62%) were positive for one of the virulence genes investigated. 48.1% (n=13) tested positive at the genes encoding for tetracycline resistance, tetA being the most prevalent one (61.5%; n=8). A high percent (33.3%; n=9) revealed the beta-lactamase (bla TEM ) resistance gene, and none of the samples tested positive for bla CMY and bla SHV genes. The genes responsible for resistance to sulfonamides (sul1) and trimethoprim (dfrA1) were detected in rates of 14.8% (n=4) and 7.4% (n=2), respectively. E. coli is highly prevalent in raw milk and unpasteurized cheeses marketed in Romania. These strains might represent an important reservoir of resistance genes which can easily spread into other European countries, given the unique market.

Abundance of antibiotic resistance genes in environmental bacteriophages.

The ecosystem is continuously exposed to a wide variety of antimicrobials through waste effluents, agricultural run-offs and animal-related and anthropogenic activities, which contribute to the spread of antibiotic resistance genes (ARGs). The contamination of ecosystems with ARGs may create increased opportunities for their transfer to naive microbes and eventually lead to entry into the human food chain. Transduction is a significant mechanism of horizontal gene transfer in natural environments, which has traditionally been underestimated as compared to transformation. We explored the presence of ARGs in environmental bacteriophages in order to recognize their contribution in the spread of ARGs in environmental settings. Bacteriophages were isolated against environmental bacterial isolates, purified and bulk cultured. They were characterized, and detection of ARG and intI genes including blaTEM, blaOXA-2, intI1, intI2, intI3, tetA and tetW was carried out by PCR. This study revealed the presence of various genes [tetA (12.7 %), intI1 (10.9 %), intI2 (10.9 %), intI3 (9.1 %), tetW (9.1 %) and blaOXA-2 (3.6 %)] and blaTEM in a significantly higher proportion (30.9 %). blaSHV, blaOXA-1, tetO, tetB, tetG, tetM and tetS were not detected in any of the phages. Soil phages were the most versatile in terms of ARG carriage. Also, the relative abundance of tetA differed significantly vis-à-vis source. The phages from organized farms showed varied ARGs as compared to the unorganized sector, although blaTEM ARG incidences did not differ significantly. The study reflects on the role of phages in dissemination of ARGs in environmental reservoirs, which may provide an early warning system for future clinically relevant resistance mechanisms.

High third-generation cephalosporin resistant Enterobacteriaceae prevalence rate among neonatal infections in Dakar, Senegal.

BackgroundNeonatal infection constitutes one of Senegal’s most important public health problems, with a mortality rate of 41 deaths per 1,000 live births.MethodsBetween January 2007 and March 2008, 242 neonates with suspected infection were recruited at three neonatal intensive care units in three major tertiary care centers in Dakar, the capital of Senegal. Neonatal infections were confirmed by positive bacterial blood or cerebrospinal fluid culture. The microbiological pattern of neonatal infections and the antibiotic susceptibility of the isolates were characterized. In addition, the genetic basis for antibiotic resistance and the genetic background of third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae were studied.ResultsA bacteriological infection was confirmed in 36.4 % (88/242) of neonates: 22.7 % (30/132) during the early-onset and 52.7 % (58/110) during the late-onset periods (p > 0.20). Group B streptococci accounted for 6.8 % of the 88 collected bacterial isolates, while most of them were Enterobacteriaceae (n = 69, 78.4 %). Of these, 55/69 (79.7 %) were 3GC-R. The bla CTX-M-15 allele, the bla SHV and the bla TEM were highly prevalent (63.5, 65.4 and 53.8 %, respectively), usually associated with qnr genes (65.4 %). Clonally related strains of 3GC-R Klebsiella pneumoniae and 3GC-R Enterobacter cloacae, the two most commonly recovered 3GC-R Enterobacteriaceae (48/55), were detected at the three hospitals, underlining the role of cross-transmission in their spread. The overall case fatality rate was 18.6 %.ConclusionsMeasures should be taken to prevent nosocomial infections and the selection of resistant bacteria.

First report of TEM-104-, SHV-99-, SHV-108-, and SHV-110-producing Klebsiella pneumoniae from Iran.

Extended-spectrum beta-lactamases (ESBLs) are bacterial enzymes capable of hydrolyzing beta-lactams. The aim of this study was to describe the prevalence of TEM- and SHV-type ESBL-producing Klebsiella pneumoniae strains in Zahedan, Southeast Iran. A total of 170 non-repetitive K. pneumoniae strains were collected from patients referred to three teaching hospitals of Zahedan. Antibiotic susceptibility testing was determined for 17 antibiotics using the Kirby-Bauer disc diffusion method. The frequency of ESBL-producing strains was calculated, and minimum inhibitory concentrations of ESBL-producing strains were determined for cefotaxime, ceftazidime, ceftriaxone, and cefpodoxime. The presence of bla TEM and bla SHV genes was tested in all ESBL-producing strains using polymerase chain reaction and DNA sequencing. Among the 170 K. pneumoniae clinical isolates, 55 (32.4%) were ESBL producers; 92.7% (n=51) and 72.7% (n=40) of the isolates carried the bla SHV and bla TEM genes, respectively, and 67.3% (n=37) carried both genes. The sequencing results showed that all bla TEM types were bla TEM-1, except for two isolates that were bla TEM-104. The bla SHV types were bla SHV-1, bla SHV-11, bla SHV-12, bla SHV-99, bla SHV-108, and bla SHV-110. The percentage of bla TEM and bla SHV among ESBL-producing K. pneumoniae isolates from Zahedan is relatively high, indicating the need for further surveillance and consideration in antibiotic use. To the best of our knowledge, this is the first report of TEM-104-, SHV-99-, SHV-108-, and SHV-110-type ESBLs among clinical isolates of K. pneumoniae from Iran, and TEM-1, SHV-1, SHV-11, and SHV-12 appear to be the dominant ESBLs in this region.

Phenotypic and Molecular Characterization of ESBL sproducing Enterobacteriaceaein A Tertiary Care Hospital

Background :There is an alarming magnitude of antibiotic resistance, production of Extended-Spectrum βLactamases (ESBLs) is a significant resistance-mechanism among Gram Negative bacteria posing challenge to the most coveted wellbeing of man. The Present study was undertaken to detect the prevalence of ESBLs producers, their pedantic antibiogram, and their genes, bla CTX-M, bla TEM &bla SHV by Multiplex PCR. Methodology:In this prospective study, 240 non repetitive Isolates of Enterobacteriaceae from various samples of different departments of Gandhi Hospital were processed for the presence of ESBL by Phenotypic screening, Jarlier DDST, Phenotypic confirmatory test (CLSI guidelines),Vitek2 System, Multiplex PCR for Molecular characterization over a period of 11⁄2 years in the Department of Microbiology Gandhi Medical College and Hospital,Secunderabad,Telangana. Results: Out 0f 240 Gram negative isolates 35.8% were phenotypically confirmed ESBLs producers. OP samples yielded 49/86(56.98%), whereas IP 37/86 (43.02%)(P value 0.0327 statistically significant).Medical and Surgical ICUs yielded 50%, Predominant organism isolated was E.coli 61.62%. All the isolates were 100% resistant to 3 rd generation Cephalosporins, 100% Sensitive to Carbapenems, Genotypic characterization was detected in 78 Isolates. Single gene was found in 73.08%. Single Predominant gene was blaCTX-M 71.9%, commonest multiple genes combination was CTX-M&SHV12.82%. Conclusion:Our study confirms global trend of resistance to beta-lactam antibiotics. ESBL strains are usually multi-drug resistant.An appropriate and judicious antibiotic use may lead to withdrawal of the selective pressure.

Hospital Effluents Are One of Several Sources of Metal, Antibiotic Resistance Genes, and Bacterial Markers Disseminated in Sub-Saharan Urban Rivers

Data concerning the occurrence of emerging biological contaminants such as antibiotic resistance genes (ARGs) and fecal indicator bacteria (FIB) in aquatic environments in Sub-Saharan African countries is limited. On the other hand, antibiotic resistance remains a worldwide problem which may pose serious potential risks to human and animal health. Consequently, there is a growing number of reports concerning the prevalence and dissemination of these contaminants into various environmental compartments. Sediments provide the opportunity to reconstruct the pollution history and evaluate impacts so this study investigates the abundance and distribution of toxic metals, FIB, and ARGs released from hospital effluent wastewaters and their presence in river sediments receiving systems. ARGs (blaTEM, blaCTX-M, blaSHV, and aadA), total bacterial load, and selected bacterial species FIB [Escherichia coli, Enterococcus (ENT)] and species (Psd) were quantified by targeting species specific genes using quantitative PCR (qPCR) in total DNA extracted from the sediments recovered from 4 hospital outlet pipes (HOP) and their river receiving systems in the City of Kinshasa in the Democratic Republic of the Congo. The results highlight the great concentration of toxic metals in HOP, reaching the values (in mg kg−1) of 47.9 (Cr), 213.6 (Cu), 1434.4 (Zn), 2.6 (Cd), 281.5 (Pb), and 13.6 (Hg). The results also highlight the highest (P < 0.05) values of 16S rRNA, FIB, and ARGs copy numbers in all sampling sites including upstream (control site), discharge point, and downstream of receiving rivers, indicating that the hospital effluent water is not an exclusive source of the biological contaminants entering the urban rivers. Significant correlation were observed between (i) all analyzed ARGs and total bacterial load (16S rRNA) 0.51 to 0.72 (p < 0.001, n = 65); (ii) ARGs (except blaTEM) and FIB and Psd 0.57 < r < 0.82 (p < 0.001, n = 65); and (iii) ARGs (except blaTEM) and toxic metals (Cd, Cr, Cu, and Zn) 0.44 to 0.72, (p < 0.001, n = 65). These findings demonstrate that several sources including hospital and urban wastewaters contribute to the spread of toxic metals and biological emerging contaminants in aquatic ecosystems.

Outbreak of Extended-Spectrum Beta-Lactamase Producing Enterobacter cloacae with High MICs of Quaternary Ammonium Compounds in a Hematology Ward Associated with Contaminated Sinks.

Objective: To investigate an outbreak of extended-spectrum beta-lactamase (ESBL) producing Enterobacter cloacae that occurred in the Hematology ward (24-bed unit) of the François Mitterrand University Hospital (Dijon, France) between January 2011 and December 2013. The outbreak involved 43 patients (10 infected and 33 colonized).Design: We performed environmental analysis to detect multiresistant E. cloacae for comparison with clinical isolates (genotyping by pulsed-field gel electrophoresis and MLST as well as ESBL-typing) and determined the MICs of the quaternary ammonium compounds (QACs) alkyldimethylbenzylammonium chloride (ADBAC) and didecyldimethylammonium chloride (DDAC). A bleach-based cleaning-disinfection program was implemented in December 2012 after mechanical removal of the biofilm in all sinks.Results: We have detected 17 ESBL-producing E. cloacae in patients sink drains, shower drains and medical sink drains. Sequencing of the bla genes performed on 60 strains recovered from patients and environment (n = 43 clinical and n = 17 environmental) revealed that bla CTX−M15 was predominant (37 isolates) followed by bla CTX−M9 plus bla SHV−12 (20 isolates). We observed a great diversity among the isolates: 14 pulsotypes (11 STs) in clinical isolates and 9 pulsotypes (7 STs) in environmental isolates. Six pulsotypes were identical between clinical and environmental isolates. MICs of the quaternary ammonium compounds widely used for disinfection were very high in clinical and environmental isolates. Immediately after the implementation of the disinfection program we noticed a substantial fall in cases number. Our findings demonstrate the role of drains as important reservoir of ESBL-producing E. cloacae and highlight the necessity to settle drains accessible to achieve correct cleaning as well as to use disinfectant with proved activity against nosocomial pathogens.

Occurrence of OXA-48 Carbapenemase and Other β-Lactamase Genes in ESBL-Producing Multidrug Resistant Escherichia coli from Dogs and Cats in the United States, 2009-2013.

Objective: The aim of this study was to explore the occurrence and molecular characterization of extended-spectrum β-lactamases (ESBL), plasmid-mediated AmpC β-lactamase (pAmpC) and carbapenemases among ESBL-producing multidrug resistant (MDR) Escherichia coli from dogs and cats in the United States.Methods: Of 2443 E.coli isolated from dogs and cats collected between August 2009 and January 2013, 68 isolates were confirmed as ESBL-producing MDR ones. PCR and sequencing were performed to identify β-lactamases and plasmid-mediated quinolone resistance (PMQR) genes, and shed light on the virulence gene profiles, phylogenetic groups and ST types.Results: Phylogenic group D and B2 accounted for 69.1% of the isolates. 50 (73.5%) isolates carried CTX-M ESBL gene, and the most predominant specific CTX-M subtype identified was blaCTX−M−15 (n = 33), followed by blaCTX−M−1 (n = 32), blaCTX−M−123 (n = 27), blaCTX−M−9 (n = 19) and blaCTX−M−14 (n = 19), and blaCTX−M−123 was firstly reported in E. coli isolates in the United States alone or in association. Other β-lactamase genes blaTEM, blaSHV, blaOXA−48, and blaCMY−2 were detected in 41.2, 29.4, 19.1, and 17.6% of 68 ESBL-producing MDR isolates, respectively. The blaTEM and blaSHV genes were classfied as ESBLs with the exception of the blaTEM−1 gene. Additionally, 42.6% (29/68) of isolates co-expressed blaCTX−M−15 and PMQR gene aac(6′)-Ib-c. The overall occurrence of virulence genes ranged from 11.8 (ireA) to 88.2% (malX), and most of virulence genes were less frequent among CTX-M-producing isolates than non-CTX-M isolates with the exception of malX and iutA. The 68 isolates analyzed were assigned to 31 STs with six being novel. Three pandemic clonal lineages ST131 (n = 10), ST648 (n = 9), and ST405 (n = 9) accounted for more than 41% of the investigated isolates, and ST648 and ST405 of phylogenetic D were firstly reported in E. coli from dogs and cats in the United States.Conclusion: blaCTX−M−123 of ESBLs and carbapenemase blaOXA−48 were firstly reported in ESBL-producing MDR E.coli from dogs and cats in the United States, and ST131, ST648, and ST405 were the predominant clonal groups.

Association of Risk Factors, Antimicrobial Resistance Trends and Occurrence of blaTEM, bla SHV and blaCTX M in Escherichia coli Causing Bacteremia.

Escherichia coli are the most frequent cause of gram negative bloodstream infection. This study was done to evaluate the association of risk factors, antimicrobial susceptibility pattern and detection of TEM, SHV and CTX M genes in the extended spectrum beta lactamase (ESBL) producing E.coli. 11,133 blood samples were processed in BacT/Alert bottles. Bacteria grown were identified and antibiotic susceptibility patterns were studied using VITEK2 system. ESBL production was tested using phenotypic method, VITEK system and by PCR. Statistical package SPSS Version 11.5 was used to do the analysis. Blood culture positive isolates were 1530 (13.7%), among which 108 were identified as Escherichia coli. The most common risk factor associated with E.coli bacteremia was diabetes and common source was UTI. E.coli were resistant to Ampicillin (86%), Piperacillin (82.4%), Ceftazidime (80.6%), Ceftriaxone (80.6%), Cefipime (77%), Aztreonam (80.6%) and Fluoroquinolones (80%). Isolates were sensitive to Carbapenems and combination drugs. ESBL was produced by 76%. CTX M bla (betalactamase) gene was common in all the ESBL isolates. ESBL producing E.coli isolates were observed to be multi drug resistant. Owing to the high prevalence of ESBL.Carbapenems are clearly the drug of choice for empirical treatment in these cases.Ertapenem may be used to prevent development of carbepenem resistant pseudomonas or acinetobacter isolates in the hospital settings. To limit the earlier development of resistance to carbepenems, it is better to de-escalate to combination drugs like piperacillin tazobactum with aminoglycosides after the susceptibility report.

IncM Plasmid R1215 Is the Source of Chromosomally Located Regions Containing Multiple Antibiotic Resistance Genes in the Globally Disseminated Acinetobacter baumannii GC1 and GC2 Clones.

Clear similarities between antibiotic resistance islands in the chromosomes of extensively antibiotic-resistant isolates from the two dominant, globally distributed Acinetobacter baumannii clones, GC1 and GC2, suggest a common origin. A close relative of the likely progenitor of both of these regions was found in R1215, a conjugative IncM plasmid from a Serratia marcescens strain isolated prior to 1980. The 37.8-kb resistance region in R1215 lies within the mucB gene and includes aacC1, aadA1, aphA1b, bla TEM, catA1, sul1, and tetA(A), genes that confer resistance to gentamicin, streptomycin and spectinomycin, kanamycin and neomycin, ampicillin, chloramphenicol, sulfamethoxazole, and tetracycline, respectively. The backbone of this region is derived from Tn1721 and is interrupted by a hybrid Tn2670 (Tn21)-Tn1696-type transposon, Tn6020, and an incomplete Tn1. After minor rearrangements, this R1215 resistance island can generate AbGRI2-0*, the predicted earliest form of the IS26-bounded AbGRI2-type resistance island of GC2 isolates, and to the multiple antibiotic resistance region (MARR) of AbaR0, the precursor of this region in AbaR-type resistance islands in the GC1 group. A 29.9-kb circle excised by IS26 has been inserted into the A.baumannii chromosome to generate AbGRI2-0*. To create the MARR of AbaR0, a different circular form, again generated by IS26 from an R1215 resistance region variant, has been opened at a different point by recombination with a copy of the sul1 gene already present in the AbaR precursor. Recent IncM plasmids related to R1215 have a variant resistance island containing a bla SHV gene in the same location. IMPORTANCE Two lineages of extensively antibiotic-resistant A.baumannii currently plaguing modern medicine each acquired resistance to all of the original antibiotics (ampicillin, tetracycline, kanamycin, and sulfonamides) by the end of the 1970s and then became resistant to antibiotics from newer families after they were introduced in the 1980s. Here, we show that, in both of the dominant globally disseminated A.baumannii clones, a related set of antibiotic resistance genes was acquired together from the same resistance region that had already evolved in an IncM plasmid. In both cases, the action of IS26 was important in this process, but homologous recombination was also involved. The findings highlight the fact that complex regions carrying several resistance genes can evolve in one location or organism and all or part of the evolved region can then move to other locations and other organisms, conferring resistance to several antibiotics in a single step.

Antimicrobial Resistance of Entrotoxigenic and Non-Entrotoxigenic Escherichia coli Isolated From Diarrheic Calves in Iran

Background: Among diarrheagenic strains of Escherichia coli, entrotoxigenic Escherichia coli (ETEC) is most commonly associated with diarrhea in calves and lambs. Broad use of antimicrobials in agriculture selects for resistant bacteria that may enter the food chain, and potentially result in foodborne disease in humans that is less responsive to treatment with conventional antibiotics. Objectives: This study was carried out to identify antimicrobial resistance in ETEC and non-ETEC isolated from diarrheic calves. Materials and Methods: Disk diffusion methods and PCR were used to detect antimicrobial resistance. Antimicrobial susceptibility testing was performed by using the standards recommended by the clinical and laboratory standard institute (CLSI). Multiplex or monoplex PCR amplification was used to identify eight antibiotic-resistant genes, including bla SHV, tet(A), Sul1, aac(3)-IV, ere(A), catA1, cmlA, aadA1 and qnr(A), which confer resistance to penicillin, tetracycline, sulfonamide, gentamicin, erythromycin, chloramphenicol, streptomycin, and fluoroquinolone, respectively. Results: Antimicrobial resistance rates for ETEC isolates were detected against penicillin (100%), tetracycline (90.9%), erythromycin (90.9%), streptomycin (90.9%), sulfonamide (63.6%), chloramphenicol (63.6%), gentamicin (45.4%) and fluoroquinolone (36.3%). Furthermore, according to the results, antimicrobial resistance for non-ETEC isolates was detected against penicillin (100%), followed by erythromycin (97.6%), tetracycline (93%), streptomycin (91.8%), sulfonamide (73.2%), chloramphenicol (51.1%), fluoroquinolone(44.1%), and gentamicin (34.8%). In addition, the distribution of the resistant genes for ETEC isolates were ere(A) (100%), catA1 (100%), cmlA (100%), aadA1 (100%), Sul1 (72.7%), tet(A) (54.5%), aac(3)-IV (54.5%), bla SHV (36.3%), and qnr(A) (9%). For non-ETEC isolates they were ere(A) (100%), aadA1 (100%), Sul1 (87.2%), catA1 (67.4%), cmlA (67.4%), tet(A) (48.8%), aac(3)-IV (48.8%), bla SHV (41.8%), and qnr(A) (3.4%). Conclusions: Among the eight antimicrobial agents examined in this investigation, the least resistance was observed against gentamicin and fluoroquinolone in both ETEC and non-ETEC isolates. Therefore, carrying out antimicrobial susceptibility tests before drug prescription seems necessary.

Risk factors for KPC-producing Klebsiella pneumoniae: watch out for surgery.

This study describes the molecular characteristics and risk factors associated with carbapenem-resistant Klebsiella pneumoniae strains. Risk factors associated with KPC-producing K. pneumoniae strains were investigated in this case-control study from May 2011 to May 2013. Bacterial identification was performed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility was determined by broth microdilution. Carbapenemase production was assessed by both modified Hodge test (MHT) and ertapenem hydrolysis using MALDI-TOF MS. The presence of β-lactamase-encoding genes was evaluated by PCR and DNA sequencing. Alterations in genes encoding K. pneumoniae outer membrane proteins were analysed by PCR and DNA sequencing as well as SDS-PAGE. Genetic relatedness among strains was determined by pulsed-field gel electrophoresis. This study included 94 patients. Longer hospitalisation, mechanical ventilation, catheters, and previous surgery were associated with KPC-producing K. pneumoniae. Sixty-eight strains showed resistance to carbapenems. Carbapenemase production was detected by MHT in 67 K. pneumoniae strains and by MALDI-TOF MS in 57. The presence of the blaKPC-2 gene was identified in 57 strains. The blaKPC-2 gene was not found in 11 carbapenem-resistant K. pneumoniae; instead, the blaCTX-M-1-like, blaCTX-M-2-like, blaCTX-M-8 like, blaCTX-M-14-like and blaSHV- like genes associated with OmpK35 and OmpK36 alterations were observed. Thirty-three KPC-producing K. pneumoniae strains were clonally related, and patients infected with these strains had a higher mortality rate (78.78 %). Our results show that KPC-producing K. pneumoniae was associated with several healthcare-related risk factors, including recent surgery.

Prevalence, risk factors and molecular epidemiology of highly resistant gram negative rods in hospitalized patients in the Dutch region Kennemerland

BackgroundThis paper describes (1) the Highly Resistant Gram Negative Rod (HR-GNR) prevalence rate, (2) their genotypes, acquired resistance genes and (3) associated risk factors of HR-GNR colonization among the hospitalized population in the Dutch region Kennemerland.MethodsBetween 1 October 2013 and 31 March 2014, cross-sectional prevalence measurements were performed in three regional hospitals as part of each hospitals infection control program. Rectal swabs were analyzed at the Regional Public Health Laboratory Kennemerland by direct culturing. Genotypes and acquired resistance genes of positive isolates were determined using Whole Genome Sequencing with the MiSeq instrument (Illumina). Association between several independent variables and HR-GNR positivity was examined using logistic regression models.ResultsOut of 427 patients, 24 HR-GNR positive isolates were recovered from 22 patients, resulting in a regional HR-GNR colonization prevalence (95 % CI) of 5.2 % (3.6–7.9). Of these 22 positive patients, 15 were Extended Spectrum Beta-Lactamase (ESBL) positive (3.5 % (2.1–5.7)), 7 patients were positive for a Fluoroquinolones and Aminoglycosides (Q&A) resistant Enterobacteriaceae (1.6 % (0.8–3.3)) and from one patient (0.2 % (0–1.3)) a Stenotrophomonas maltophilia resistant towards co-trimoxazole was isolated. No carbapenemase producing Enterobacteriaceae (CPE), multi-resistant Acinetobacter species or multi-resistant Pseudomonas aeruginosa were isolated. The ESBL genes found were blaCTX-M-1 (n = 4, 25.0 %), blaCTX-M-15 (n = 3, 18.8 %), blaCTX-M-27 (n = 2, 12.5 %), blaCTX-M-14b (n = 2, 12.5 %), blaCTX-M-9 (n = 2, 12.5 %), blaCTX-M-14 (n = 1, 6.3 %), blaCTX-M-3 (n = 1, 6.3 %), blaSHV-11 (n = 1, 6.3 %) and blaSHV-12 (n = 1, 6.3 %). Being known HR-GNR positive in the past was the only significant associated risk factor for HR-GNR positivity, odds ratio (95 % CI): 7.32 (1.82–29.35), p-value = 0.005.ConclusionsSimilar ESBL prevalence rates and genotypes (3.5 %) were found in comparison to other Dutch studies. When previously HR-GNR positive patients are readmitted, they should be screened for HR-GNR colonization since colonization with GR-GNRs could be prolonged. We recommend for future studies to include all defined HR-GNRs in addition to ESBLs in prevalence studies, in order to obtain a more comprehensive overview of colonization with HR-GNRs.

Prevalence of plasmid-mediated multidrug resistance determinants in fluoroquinolone-resistant bacteria isolated from sewage and surface water.

Fluoroquinolones (FQs) are fully synthetic broad-spectrum antibacterial agents that are becoming increasingly popular in the treatment of clinical and veterinary infections. Being excreted during treatment, mostly as active compounds, their biological action is not limited to the therapeutic site, but it is moved further as resistance selection pressure into the environment. Water environment is an ideal medium for the aggregation and dissemination of antibiotics, antibiotic-resistant bacteria (ARB), and antibiotic resistance genes (ARGs), which can pose a serious threat to human health. Because of this, the aim of this study was to determine the number of fluoroquinolone-resistant bacteria (FQRB) and their share in total heterotrophic plate counts (HPC) in treated wastewater (TWW), and upstream and downstream river water (URW, DRW) samples where TWW is discharged. The spread of plasmid-mediated quinolone resistance (PMQR) determinants and the presence/absence of resistance genes to other most popular antibiotic groups (against tetracyclines and beta-lactams) in selected 116 multiresistant isolates were investigated. The share of FQRB in total HPC in all samples was rather small and ranged from 0.7 % in URW samples to 7.5 % in TWW. Bacteria from Escherichia (25.0 %), Acinetobacter (25.0 %), and Aeromonas (6.9 %) genera were predominant in the FQRB group. Fluoroquinolone resistance was mostly caused by the presence of the gene aac(6′)-1b-cr (91.4 %). More rarely reported was the occurrence of qnrS, qnrD, as well as oqxA, but qnrA, qnrB, qepA, and oqxB were extremely rarely or never noted in FQRB. The most prevalent bacterial genes connected with beta-lactams’ resistance in FQRB were blaTEM, blaOXA, and blaCTX-M. The blaSHV was less common in the community of FQRB. The occurrence of bla genes was reported in almost 29.3 % of FQRB. The most abundant tet genes in FQRB were tet(A), tet(L), tet(K), and tet(S). The prevalence of tet genes was observed in 41.4 % of FQRB. The highest prevalence of multidrug-resistant (MDR) microorganisms was detected in TWW and DRW samples. It indicates that discharged TWW harbors multiresistant bacterial strains and that mobile PMQR and ARGs elements may have a selective pressure for species affiliated to bacteria in the river water.Electronic supplementary materialThe online version of this article (doi:10.1007/s11356-016-6221-4) contains supplementary material, which is available to authorized users.

Prevalence and characterization of extended-spectrum beta-lactamases producing Enterobacteriaceae in healthy children and associated risk factors.

BackgroundCommunity acquired infections due to extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) had been increased. The fecal flora of children in the community represents a huge potential reservoir for ESBLs which are located on highly transmissible plasmids. This study examined the prevalence of ESBL-PE fecal carriage, antimicrobial susceptibility pattern, possible risk factors, and characterized the genes encoding these ESBL enzymes in Lebanese children community. MethodsA total of 125 rectal swabs were taken from healthy children aged from 1 to 5 years. Detection of ESBLs was carried out using combination-disc method test and multiplex PCR. A questionnaire concerning child’s lifestyle and risk factors for ESBL carriage was illustrated.ResultsThirty-one of 125 participants (24.8 %) carried ESBL-PE. Regular consumption of meat, and chicken were significantly associated with high carriage rate of ESBL-PE, while dairy products (milk, yogurt, cheese) association was non-significant. Intimate hygiene habits were found also affecting the carriage rate. Multiple bla genes were the most common, 48.4 % (15/31) of ESBL-PE carried both blaCTX-M and blaTEM, and 22.6 % (7/31) carried blaCTX-M, blaSHV, and blaTEM, 29 % (9) carried blaCTX-M only. Concerning CTX-M-types, CTX-M-9 was the most predominant (24/31) and mostly in combination with CTX-M-15 type.ConclusionHigh rate of colonization in healthy children with ESBL-PE was observed, regular consumption of dietary products from animal source (meat or chicken) were associated with this colonization in the community in non-hospitalized children. To our best knowledge it is the first study about regular consumption of dairy product as a risk factor for ESBL-PE community carriage, the first data about the carriage rate of ESBL-PE in community children in Lebanon and Middle East, and for the wide dissemination of CTX-M-9 type in this population.

Quinolone Resistance among Extended-Spectrum β-Lactamases Producing Klebsiella Pneumoniae in Sohag University Hospital, Upper Egypt

Background: Extended-spectrum β-lactamases (ESBLs) production is the major resistance mechanism to β-lactam antibiotics in Enterobacteriaceae. Moreover, the emergence of quinolone resistance in ESBLs-producing isolates had become a global threat for treatment of infections caused by these bacteria. Objectives: We investigated the association between ESBLs production and quinolone resistance in Klebsiella pneumoniae isolates. Methodology: A total of 148 isolates of K. pneumoniae collected from Sohag University Hospital over the period of 10 months between June 2014 and April 2015. Identification of Klebsiella pneumoniae was performed with the Vitek 2 system (bioMerieux, France). Screening for extended spectrum beta lactamases (ESBLs) production was done with the Vitek 2 system (bioMerieux, France) and confirmed using the combined disk diffusion test (CDDT) .The ESBL’s genotype was then analyzed by multiplex PCR of bla TEM , bla SHV and bla CTX-M genes. Also multiplex PCR was used for detection of quinolone resistance determinant genes qnrA, qnrB and qnrS among ESBLs-producing K. pneumonia isolates. Results : In our study we found that among the 148 K. pneumoniae isolates; 121 (81.7%) were ESBLs producers according to CDDT results, bla TEM was found in 44% of them, while bla CTX-M found in 6%, bla TEM and bla CTX-M together found in 39% of the isolates, but in 7% bla TEM , bla SHV were found together, the three genes bla TEM , bla SHV and bla CTX-M were found in 4% but none of the isolates carried bla SHV gene alone or with bla CTX-M . 113(93%) of the 121 ESBLs producing isolates were found to carry one or more of quinolone resistance (QNR) genes qnrA, qnrB, qnrS. The three genes together were found in (53.9%) of the isolates, while qnrS was found in (29.1%), qnrA in (11%) of the isolates and (6%) carried both qnr B and S. Conclusions: Our results showed coexistence of ESBLs production and quinolone resistance in the majority of the K. pneumoniae isolates suggesting that more care should be taken for the choice of antibiotic therapy beside strict application of infection control policies to prevent dissemination of the multidrug resistant strains.

Detection of Extended Spectrum Beta-Lactamases Resistance Genes among Bacteria Isolated from Selected Drinking Water Distribution Channels in Southwestern Nigeria.

Extended Spectrum Beta-Lactamases (ESBL) provide high level resistance to beta-lactam antibiotics among bacteria. In this study, previously described multidrug resistant bacteria from raw, treated, and municipal taps of DWDS from selected dams in southwestern Nigeria were assessed for the presence of ESBL resistance genes which include bla TEM, bla SHV, and bla CTX by PCR amplification. A total of 164 bacteria spread across treated (33), raw (66), and municipal taps (68), belonging to α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Flavobacteriia, Bacilli, and Actinobacteria group, were selected for this study. Among these bacteria, the most commonly observed resistance was for ampicillin and amoxicillin/clavulanic acid (61 isolates). Sixty-one isolates carried at least one of the targeted ESBL genes with bla TEM being the most abundant (50/61) and bla CTX being detected least (3/61). Klebsiella was the most frequently identified genus (18.03%) to harbour ESBL gene followed by Proteus (14.75%). Moreover, combinations of two ESBL genes, bla SHV + bla TEM or bla CTX + bla TEM, were observed in 11 and 1 isolate, respectively. In conclusion, classic bla TEM ESBL gene was present in multiple bacterial strains that were isolated from DWDS sources in Nigeria. These environments may serve as foci exchange of genetic traits in a diversity of Gram-negative bacteria.

Detection of ESBL Producing Bacteria in Cases of Urinary Tract Infection in Pediatric Department at Benha University Hospital

Background: Extended-spectrum beta-lactamases (ESBLs) are rapidly evolving group of beta-lactamase enzymes commonly produced by the Gram negative bacteria. Objectives: This work aimed to detect the bacterial pathogens that cause urinary tract infection and to detect ESBL producing bacteria among them in Pediatric Department at Benha University Hospital. Methodology: This study was conducted on 118 children patients suffering from UTI collected from Outpatient and Inpatient of pediatric department at Benha University during the period from January 2015 to the end of July 2015. They were 77 girls and 41 boys and their ages ranged from 1 to 13 years.118 Urine samples were subjected to isolation and identification of causative organisms, screened for ESBL production by the pattern of antimicrobial susceptibilities. Phenotypic identification for ESBL production was confirmed by double disc diffusion test, bla SHV and bla CTX-M genes in ESBL, producing isolates were detected by using multiplex PCR. Results: This study showed that the most common cause of children UTI was E.coli (55.1%) followed by klebsiella pneumoniae (21.2%). Thirty seven (31.4%) of 118 bacterial isolates that tested by double disc confirmatory test were ESBL positive. Among the ESBL producing bacteria, 16 out of 37 (43.24%) have CTX-m and SHV genes together while 3 out of 37 (8.1%) have SHV gene only. Conclusion: Our findings showed that majority of ESBL isolates were positive for both CTXm and SHV genes (43.24%) but SHV gene alone was positive only in (8.1%) of isolates, So studies should be done in different regions of Egypt to find the common ESBL genes present in that geographical area for epidemiological purposes. Also ESBL producing isolates have been increasingly recognized in hospitals in Egypt and are associated with multiple drug resistance. Thus, ESBL producing organisms should be identified quickly so that appropriate antibiotic usage and infection control measures can be implemented.

Prevalence and molecular epidemiology of extended spectrum beta-lactamase producing Escherichia coli from hospital and community settings in Egypt

Article history: Received on: 13/11/2015 Revised on: 20/12/2015 Accepted on: 04/01/2016 Available online: 26/01/2016 The prevalence and molecular epidemiology of Escherichia coli that produce extended spectrum β-lactamase (ESBL) in Cairo, Egypt was investigated. Ninety E. coli isolates were collected along the period of September to November 2012 from hospital and community settings. Antibiotic susceptibility of the E. coli isolates was determined by disk diffusion method. All isolates were screened phenotypically for ESBL production by combination disk method. The presence of bla CTX-M-I, bla CTX-M-IV, bla TEM and bla SHV genes in ESBLproducing E. coli was examined by PCR and sequencing experiments. The results showed high prevalence of ESBL-producing E. coli, 52% of the collected isolates were ESBL producers. The ESBL-producing isolates significantly (P < 0.05) had increased resistance compared with non–ESBL producers to cefuroxime, cefotaxime, ceftazidime, cefepime, ciprofloxacin, and co-trimoxazole. Imipenem was the most effective drug against ESBL producing isolates. All ESBL producing E. coli isolates were multi drug resistant (MDR) to eight antibiotics or more. Detection of ESBL genes in selected MDR-ESBL producing E. coli revealed that bla CTX-M-I was the most prevalent ESBL type. It is clear that the prevalence of ESBL producing E. coli in Cairo, Egypt is alarming high. This study is useful for clinician in order to improve the empiric treatment.