Enterobacteriaceae producing OXA-48-like carbapenemases in Poland, 2013-January 2017.

Objectives: To analyse OXA-48 (OXA-48/181)-type carbapenemase-producing Enterobacteriaceae reported in Poland from 2013 until January 2017. Methods: Bacterial isolates were typed by PFGE and MLST. Genes coding for OXA-48/181 types and other ß-lactamases were amplified and sequenced. Mobile elements with blaOXA-48/181-like genes were PCR mapped. blaOXA-48/181-carrying plasmids were characterized by nuclease S1-hybridization profiling, transfer assays and PCR-based replicon typing, while the chromosomal location of the genes was confirmed by the I-CeuI analysis. Results: Fifty-four isolates from 52 patients in 20 hospitals (14 cities) were included, in 14 cases having probable foreign origins indicated. The organisms were genetically diverse and represented numerous pandemic clones, including Klebsiella pneumoniae ST395 (n = 23), ST11, ST15 and ST101, Escherichia coli ST38, ST410 and ST648, and Enterobacter cloacae complex ST78. These produced OXA-48 (n = 49), OXA-181 (n = 4) or OXA-232 (n = 1). One of five K. pneumoniae ST395 pulsotypes caused a multicentre outbreak with 18 cases, which significantly contributed to the total number of patients. Depending on the variant, the blaOXA-48/181-like genes were parts of the Tn1999-, Tn2013- or Tn2016-like transposons, with blaOXA-48 found in an ISEcp1-associated module (Tn2016-like) for the first time. Three genotypes, including E. coli ST38, had chromosomal blaOXA-48 genes, while others carried transmissible IncL (∼60 kb; blaOXA-48; n = 30), IncM (∼80-95 kb; blaOXA-48; n = 4), IncX3 (∼50 kb; blaOXA-181; n = 4) or non-typeable (∼90-160 kb; blaOXA-48 or blaOXA-232) plasmids. Conclusions: Even though OXA-48/181 producers seem to occur infrequently in Poland, their epidemiology has been marked by various phenomena, namely multiple imports, several limited transmissions plus one larger clonal outbreak, and possible plasmid transfers.

NDM-producing Enterobacteriaceae in Poland, 2012-14: inter-regional outbreak of Klebsiella pneumoniae ST11 and sporadic cases.

OBJECTIVES: The objective of this study was to characterize New Delhi metallo-ß-lactamase (NDM)-producing Enterobacteriaceae isolates reported in Poland in 2012-14. METHODS: Representative isolates were typed by PFGE and MLST. NDM and other ß-lactamase genes were amplified and sequenced. Plasmids with blaNDM genes were analysed by nuclease S1 plus hybridization profiling, by transfer assays and by PCR-based replicon typing. The blaNDM genetic context was studied by PCR mapping assays. RESULTS: Of 374 cases of infection/colonization with NDM-positive Enterobacteriaceae identified in 2012-14, 370 cases in 40 hospitals, 10 outpatient clinics and 1 nursing home were associated with a Klebsiella pneumoniae outbreak with epicentres in Poznan and Warsaw. The outbreak strain of K. pneumoniae ST11 was similar to an isolate from the Czech Republic from 2013. Like the Czech strain, many of the isolates had two blaNDM-1-carrying IncFII- and IncR-type plasmids of variable size, sharing a blaNDM-1-containing segment. The early isolates also produced CTX-M-15 co-encoded by the IncR-type plasmids, and differentiated later by extensive plasmid rearrangements. Four other NDM cases were reported in 2013, three being associated with arrivals from Montenegro, India or Afghanistan. The Indian Escherichia coli ST448 NDM-5 isolate revealed similarity to a recent isolate from Spain, including the blaNDM genetic context observed previously in E. coli strains in Poland and France (of Congolese and Indian origins, respectively). The Afghani Proteus mirabilis was the second isolate of this species with a chromosomal blaNDM-1 location. CONCLUSIONS: The largest NDM outbreak in a non-endemic country has been observed, being an alarming phenomenon in resistance epidemiology in Poland.

KPC-Like Carbapenemase-Producing Enterobacteriaceae Colonizing Patients in Europe and Israel.

In a 2008-2011 survey, 17,945 patients in 18 hospital units in Europe and Israel were screened for carriage of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae, resulting in identification of 124 positive patients. The isolates were dominated by Klebsiella pneumoniae sequence type 258 (ST258) KPC-2 and ST512 KPC-3, mainly from Greece and Italy, respectively, whereas Israeli isolates were of diverse species, clones, and KPC variants. Various blaKPC platforms were observed, among which IncFIIK-FIBK plasmids with blaKPC-2/-3 genes in the Tn4401a transposon prevailed.

Phylogenetic lineages, clones and ß-lactamases in an international collection of Klebsiella oxytoca isolates non-susceptible to expanded-spectrum cephalosporins.

OBJECTIVES: The objective of this study was to examine Klebsiella oxytoca clonal and phylogenetic diversity, based on an international collection of carriage isolates non-susceptible to expanded-spectrum cephalosporins (ESCs). METHODS: The study material comprised 68 rectal carriage K. oxytoca isolates non-susceptible to ESCs recovered in 2008-11 from patients in 14 hospitals across Europe and Israel. ESC resistance was tested phenotypically; genes encoding ESBLs, AmpC cephalosporinases and carbapenemases were amplified and sequenced. The isolates were typed by PFGE and MLST, followed by sequencing of blaOXY genes. RESULTS: MLST and PFGE distinguished 34 STs and 47 pulsotypes among the isolates, respectively. Six STs were split into several pulsotypes each. Five STs were more prevalent (n = 2-9) and occurred in several countries each, including ST2, ST9 and ST141, which belong to a growing international clonal complex (CC), CC2. Four phylogenetic lineages were distinguished, each with another type of chromosomal OXY-type ß-lactamase. Three of these, with OXY-1/-5, OXY-2 types and OXY-4, corresponded to previously described phylogroups KoI, KoII and KoIV, respectively. A single isolate from Israel represented a distinct lineage with a newly defined OXY-7 type. The phylogroups showed interesting differences in mechanisms of ESC resistance; KoI strains rarely overexpressed the OXY enzymes but commonly produced ESBLs, whereas KoII strains often were OXY hyperproducers and carried ESBLs much less frequently. AmpCs (DHA-1) and carbapenemases (VIM-1) occurred sporadically. CONCLUSIONS: The study confirmed the high genetic diversity of the collection of K. oxytoca ESC-non-susceptible isolates, composed of phylogroups with distinct types of OXY-type ß-lactamases, and revealed some STs of broad geographical distribution.

Survey of metallo-ß-lactamase-producing Enterobacteriaceae colonizing patients in European ICUs and rehabilitation units, 2008-11.

OBJECTIVES: The objective of this study was to perform a multinational survey of patients' colonization by metallo-ß-lactamase (MBL)-producing Enterobacteriaceae, including their molecular characterization. METHODS: Patients in 18 hospital units across Europe and Israel (n = 17 945) were screened between mid-2008 and mid-2011. MBL-producing isolates were typed by PFGE and MLST. MBL genes were amplified and sequenced within their integrons. Plasmids with MBL genes were analysed by nuclease S1 plus hybridization profiling, mating and transformation assays, and by PCR-based replicon typing. RESULTS: Ninety-one patients in nine centres (six countries), including 62 patients in two Greek ICUs, carried 94 non-duplicate MBL-producing organisms. Klebsiella pneumoniae isolates from Greece dominated (n = 57) and belonged mainly to ST147, ST36 and ST383. All but one of the isolates expressed VIM-1-type MBLs. Isolates of Greek origins produced five enzymes, including new VIM-39, encoded by class 1 integrons of four types. In-e541-like elements prevailed, comprising six variants located on IncR, IncFIIK, IncR + FIIK, IncR + A/C or non-typeable plasmids. The other group were new In4873 and In4863, being the first In416-like elements identified in Greece, which were present on IncA/C or non-typeable plasmids. Isolates from other countries produced only VIM-1 and the major integron was In916, identified in 16 organisms from France, Italy and Spain. In916 was carried by four plasmid types, including IncA/C, IncFIIK and IncHI2. Other integrons included a new element, In3103, in Spain and In110 identified only in Latvia. CONCLUSIONS: This study provided fully comparable data on the occurrence and molecular characteristics of VIM-producing Enterobacteriaceae in a group of hospital units across Europe, documenting recent changes in their epidemiology.

MLST reveals potentially high-risk international clones of Enterobacter cloacae.

OBJECTIVES: To perform the first multinational Enterobacter cloacae clonality study, using the MLST scheme newly developed in Japan. METHODS: The analysis included 195 rectal carriage E. cloacae isolates resistant to expanded-spectrum cephalosporins (ESCs), collected from patients in 12 hospital units across Europe and Israel. All of the isolates were typed by PFGE and 173 isolates were subjected to MLST. ESC resistance was analysed phenotypically; genes encoding ESBLs and carbapenemases were identified by PCR and sequencing. RESULTS: MLST distinguished 88 STs, which correlated with the PFGE data. PFGE was more discriminatory, producing 129 pulsotypes (169 patterns). Numerous STs were observed in several countries each. The most widespread were ST66, ST78, ST108 and ST114, each having at least 10 isolates from three to five countries, diversified into multiple pulsotypes, with clusters of related isolates in one or more centres. Analysis of the STs against the MLST database revealed several epidemic clonal complexes, such as those with central genotypes ST74 (including ST78) or ST114 (including ST66). ESC resistance was equally related to overexpression of the AmpC cephalosporinase and to ESBL production. Among ESBL producers some spreading subclones were identified, including specific ST66, ST78 and ST114 pulsotypes, associated with CTX-M-15 production. Several isolates produced carbapenemase VIM-1 or KPC-2. CONCLUSIONS: Together with the information available in the MLST database, our results suggest that, like Escherichia coli and Klebsiella pneumoniae, E. cloacae harbours clonal lineages of increased epidemic potential that may be associated with resistance spread.

A multinational study of colonization with extended spectrum ß-lactamase-producing Enterobacteriaceae in healthcare personnel and family members of carrier patients hospitalized in rehabilitation centres.

The study aims were: (i) to define the prevalence of and risk factors for colonization by extended spectrum ß-lactamase (ESBL) -producing Enterobacteriaceae (EPE) among healthcare workers (HCWs) and family members (FMs) of EPE-colonized patients in rehabilitation units and (ii) to compare EPE isolates from these three groups. The study included 286 FMs of 194 EPE-carrying patients identified in five rehabilitation units located in Israel, Italy, France and Spain. The EPE were detected in rectal swabs from 26 (9%) of 286 FMs screened. In multivariate analyses, older age of FM, greater mean number of hours spent with the patient, being a daughter or a female spouse of a patient, and chronic lung disease of the patient were significantly associated with carriage in the FM. Escherichia coli was the most common organism (76%), followed by Klebsiella pneumoniae (19%). Isolates were typed by pulsed field gel electrophoresis and multilocus sequence typing, and ESBLs were identified by PCR sequencing. A comparison of paired species isolates from FMs and their respective patient showed that 17 of 23 strains were indistinguishable. EPE were detected in 35 (3.5%, E. coli = 34) of the 1001 HCWs screened. Feeding patients was associated with EPE carriage by HCWs. Only 7 of 23 E. coli subclones cultured from HCWs were also represented among 376 patient-derived ESBL-producing E. coli isolates from the same rehabilitation units. In Spain, a higher proportion of HCWs and FMs were ESBL carriers than elsewhere (p <0.05). In conclusion, the molecular and epidemiological data suggest that FMs are at higher risk of EPE acquisition from their relative patients than HCWs.

The first NDM metallo-ß-lactamase-producing Enterobacteriaceae isolate in Poland: evolution of IncFII-type plasmids carrying the bla(NDM-1) gene.

Poland's first Enterobacteriaceae isolate producing the New Delhi metallo-ß-lactamase (NDM) was identified in August 2011. Escherichia coli sequence type ST410 NDM-1 was cultured from a critically ill patient who had been transferred directly from the Congo. The blaNDM-1 gene was carried by conjugative IncFII-type plasmid pMC-NDM (87,619 bp), which showed structural similarity to plasmid pGUE-NDM, which was identified earlier in France in an E. coli ST131 isolate of Indian origin.

Comparative population analysis of Klebsiella pneumoniae strains with extended-spectrum ß-lactamases colonizing patients in rehabilitation centers in four countries.

The international project MOSAR was conducted in five rehabilitation centers; patients were screened for rectal carriage of extended-spectrum ß-lactamase (ESBL)-producing members of the Enterobacteriaceae. Among 229 Klebsiella pneumoniae isolates, four clonal groups (CG) or complexes (CC) prevailed: CG17 in France, CG101 in Italy, CG15 in Spain, and CC147 in Israel. ESBLs, mainly CTX-Ms, were produced by 226 isolates; three isolates expressed AmpC-like cephalosporinases. High genetic diversity of K. pneumoniae populations was observed, with specific characteristics at each center.

Clonal structure, extended-spectrum ß-lactamases, and acquired AmpC-type cephalosporinases of Escherichia coli populations colonizing patients in rehabilitation centers in four countries.

The prospective project MOSAR was conducted in five rehabilitation units: the Berck Maritime Hôpital (Berck, France), Fondazione Santa Lucia (Rome, Italy), Guttmann Institute (GI; Barcelona, Spain), and Loewenstein Hospital and Tel-Aviv Souraski Medical Center (TA) (Tel-Aviv, Israel). Patients were screened for carriage of Enterobacteriaceae resistant to expanded-spectrum cephalosporins (ESCs) from admission until discharge. The aim of this study was to characterize the clonal structure, extended-spectrum ß-lactamases (ESBLs), and acquired AmpC-like cephalosporinases in the Escherichia coli populations collected. A total of 376 isolates were randomly selected. The overall number of sequence types (STs) was 76, including 7 STs that grouped at least 10 isolates from at least three centers each, namely, STs 10, 38, 69, 131, 405, 410, and 648. These clones comprised 65.2% of all isolates, and ST131 alone comprised 41.2%. Of 54 STs observed only in one center, some STs played a locally significant role, like ST156 and ST393 in GI or ST372 and ST398 in TA. Among 16 new STs, five arose from evolution within the ST10 and ST131 clonal complexes. ESBLs and AmpCs accounted for 94.7% and 5.6% of the ESC-hydrolyzing ß-lactamases, respectively, being dominated by the CTX-M-like enzymes (79.9%), followed by the SHV (13.5%) and CMY-2 (5.3%) types. CTX-M-15 was the most prevalent ß-lactamase overall (40.6%); other ubiquitous enzymes were CTX-M-14 and CMY-2. Almost none of the common clones correlated strictly with one ß-lactamase; although 58.7% of ST131 isolates produced CTX-M-15, the clone also expressed nine other enzymes. A number of clone variants with specific pulsed-field gel electrophoresis and ESBL types were spread in some locales, potentially representing newly emerging E. coli epidemic strains.

A binational cohort study of intestinal colonization with extended-spectrum ß-lactamase-producing Proteus mirabilis in patients admitted to rehabilitation centres.

The aims of our study were to analyse the risk factors for colonization by Extended-spectrum ß-lactamases (ESBL)-producing Proteus mirabilis (ESBL-PM) in rehabilitation patients and to characterize the molecular features of these strains. The study was conducted in two rehabilitation centres located in Rome, Italy (Fondazione Santa Lucia IRCCS (FSL)), and Tel-Aviv, Israel (Tel-Aviv Sourasky Medical Center (TASMC)). Carriage of ESBL-PM was surveyed by rectal swabs. Strain typing was performed by pulsed-field gel electrophoresis (PFGE). Identification of ESBL genes was done by PCR and sequencing. Patients admitted to the same institutions without ESBL carriage were included as controls. The study group included 70 and 41 patients from FSL and TASMC, respectively. In FSL, the multivariate analysis identified severe acute brain injury (OR = 15, 95% CI = 3.2-69.5, p 0.001), decubitus ulcer (OR = 3.5, 95% CI = 1.2-9.8, p 0.018) and recent treatment with quinolones (OR = 5.7, 95% CI = 1.07-30.1, p 0.042) as independent risk factors. ESBL-PM carriers stayed longer in the hospital on average and were less likely to be discharged home. No significant risk factor was identified in TASMC. There were no similarities in PFGE types or ESBL genes between the ESBL-PM isolates from the two institutions. In both hospitals, a variety of PFGE types existed but a single ESBL type predominated, namely TEM-92 in FSL (n = 64/70; 91%) and CTX-M-2 in TASMC (n = 37/41; 90%). A new TEM ESBL variant, TEM-177 was identified in FSL. The clonal diversity and the predominance of a single ESBL type suggested that horizontal gene transfer played an important role in dissemination of resistance. The development of a population analysis tool that would allow tracing deeper genetic relationships is required.

Transmission dynamics of ESBL-producing Escherichia coli clones in rehabilitation wards at a tertiary care centre.

Increasing resistance due to the production of ESBL in Escherichia coli (ESBL-E. coli) has become a major threat to public health. Our aims were to study the incidence of ESBL-E. coli acquisition during hospitalization and the transmission rates of different ESBL-E. coli clones. This was a prospective case-control study, conducted in two geriatric rehabilitation wards in Tel-Aviv. Serial rectal cultures were collected from admission till discharge. All patient-unique ESBL-E. coli isolates were subjected to molecular typing by PFGE, MLST and determination of ESBL genes. An acquisition of ESBL-E. coli was defined as traceable when a patient with the same ST, PFGE type and ESBL gene was hospitalized in the same ward in parallel to the acquisition case. ESBL-E. colis were recovered from 125 patients out of 492 enrolled: 52 were recovered upon admission, 59 acquired ESBL-E. coli during their stay, and there was undetermined status in 14 patients. A low Norton's score was associated with acquisition (O.R. 1.14 for each point, 95% C.I. 1.01-1.29, p < 0.05). ESBL-E. coli infections (n = 9) had occurred only in ESBL-E. coli carriers. The pandemic ST131 clone was the most common (48/125). The majority of the isolates (101/125) produced CTX-M-type ESBL. Of the 59 acquisition cases, 32 were traced to another patient. In-hospital dissemination was highest in the CTX-M-27-producing ST131 and the SHV-5-producing ST372 sub-clones (acquisition/admission ratios of 17/11 and 9/3, respectively), with almost all cases traced to other patients. In conclusion, most ESBL-E. coli acquisition cases were traceable to other patients. The transmission potential varied significantly between ESBL-E. coli clones.

Evolution and spread of a multidrug-resistant Proteus mirabilis clone with chromosomal AmpC-type cephalosporinases in Europe.

Proteus mirabilis isolates obtained in 1999 to 2008 from three European countries were analyzed; all carried chromosomal AmpC-type cephalosporinase bla(CMY) genes from a Citrobacter freundii origin (bla(CMY-2)-like genes). Isolates from Poland harbored several bla(CMY) genes (bla(CMY-4), bla(CMY-12), bla(CMY-14), bla(CMY-15), and bla(CMY-38) and the new gene bla(CMY-45)), while isolates from Italy and Greece harbored bla(CMY-16) only. Earlier isolates with bla(CMY-4) or bla(CMY-12), recovered in France from Greek and Algerian patients, were also studied. All isolates showed striking similarities. Their bla(CMY) genes resided within ISEcp1 transposition modules, named Tn6093, characterized by a 110-bp distance between ISEcp1 and bla(CMY), and identical fragments of both C. freundii DNA and a ColE1-type plasmid backbone. Moreover, these modules were inserted into the same chromosomal site, within the pepQ gene. Since ColE1 plasmids carrying ISEcp1 with similar C. freundii DNA fragments (Tn6114) had been identified earlier, it is likely that a similar molecule had mediated at some stage this DNA transfer between C. freundii and P. mirabilis. In addition, isolates with bla(CMY-12), bla(CMY-15), and bla(CMY-38) genes harbored a second bla(CMY) copy within a shorter ISEcp1 module (Tn6113), always inserted downstream of the ppiD gene. Sequence analysis of all mobile bla(CMY-2)-like genes indicated that those integrated in the P. mirabilis chromosome form a distinct cluster that may have evolved by the stepwise accumulation of mutations. All of these observations, coupled to strain typing data, suggest that the bla(CMY) genes studied here may have originated from a single ISEcp1-mediated mobilization-transfer-integration process, followed by the spread and evolution of a P. mirabilis clone over time and a large geographic area.

Resistance patterns of selected respiratory tract pathogens in Poland.

This study presents the results of a survey of the in-vitro susceptibility to antimicrobial agents of major pathogens responsible for community-acquired respiratory tract infections in Poland during 2002-2004. The collection of 1184 bacterial isolates comprised 398 Streptococcus pneumoniae, 344 Haemophilus influenzae, 302 Streptococcus pyogenes and 140 Moraxella catarrhalis. Among the pneumococcal isolates, 16.8% were penicillin-non-susceptible (PNSP), of which 80.6% were identified as multidrug-resistant. Overall, 9.0% of H. influenzae isolates were beta-lactamase-positive, although this percentage increased noticeably in the third year of the study. Based on PCR results, 12.8% of H. influenzae isolates were identified as low-level beta-lactamase-negative, ampicillin-resistant (BLNAR), and one isolate as low-level beta-lactamase-positive, amoxycillin-clavulanic acid-resistant (BLPACR). Pulsed-field gel electrophoresis (PFGE) classified 45 H. influenzae isolates with altered penicillin-binding proteins into 15 PFGE types, including two predominant types (with four and six sub-types) containing 15 and ten isolates, respectively. Resistance to tetracycline, erythromycin and clindamycin was found in 20.9%, 8.9% and 4.6% of S. pyogenes isolates, respectively. The production of beta-lactamase characterised 91.4% of M. catarrhalis isolates. In summary, the overall occurrence of PNSP in Poland remains stable, although there was a noticeable increase in the proportion of fully-resistant isolates. A rising trend in the prevalence of beta-lactamase producers and low-level BLNAR isolates was observed among Polish isolates of H. influenzae.

Selection of a teicoplanin-resistant Enterococcus faecium mutant during an outbreak caused by vancomycin-resistant enterococci with the vanB phenotype.

Vancomycin-resistant enterococci (VRE) have recently become an increasing problem in hospitals in Poland, being responsible for a growing number of nosocomial outbreaks. In this work, we have analyzed the second outbreak of VRE with the VanB phenotype to be identified in the country. It was caused by clonal dissemination of a single strain of vancomycin-resistant Enterococcus faecalis (VRES) and horizontal transmission of vancomycin resistance genes among several vancomycin-resistant Enterococcus faecium (VREM) strains. Two similar restriction fragment length polymorphism types of the vanB gene cluster characterized VRES and VREM isolates, and they both contained the same vanB2 variant of the vanB gene. Two vancomycin-susceptible E. faecium (VSEM) isolates, recovered from the same wards during the outbreak, proved to be related to certain VREM isolates and could represent endemic strains that had acquired vancomycin resistance. One VSEM and four VREM isolates, all identified in the same patient, belonged to a single clone, although they revealed remarkable diversity in terms of susceptibility, PFGE patterns, plasmid content, and number of vanB gene cluster copies. Most probably they reflected the dynamic evolution of an E. faecium strain in the course of infection of a single patient. One of the VREM isolates turned out to be resistant to teicoplanin, which coincided with the use of this antibiotic in the patient's therapy. Its vanB gene variant differed by a single mutation from that found in other isolates; however, it also lacked a large part of the vanB gene cluster, including the regulatory genes vanR(B) and -S(B), and the vancomycin-inducible promoter P(YB). Expression of the resistance genes vanH(B), -B, and -X(B) was constitutive in the mutant, and this phenomenon was responsible for its unusual phenotype.

A novel complex mutant beta-lactamase, TEM-68, identified in a Klebsiella pneumoniae isolate from an outbreak of extended-spectrum beta-lactamase-producing Klebsiellae.

Twenty-two Klebsiella pneumoniae and two K. oxytoca extended-spectrum beta-lactamase (ESBL)-producing isolates were collected in 1996 from patients in two pediatric wards of the University Hospital in Wroclaw, Poland. Molecular typing has revealed that the K. pneumoniae isolates represented four different epidemic strains. Three kinds of enzymes with ESBL activity (pI values of 5.7, 6.0, and 8.2) were identified. The pI 6.0 beta-lactamases belonged to the TEM family, and sequencing of the bla(TEM) genes amplified from representative isolates revealed that these enzymes were TEM-47, previously identified in K. pneumoniae isolates from pediatric hospitals in Lódz and Warsaw. One of the TEM-47-producing strains from Wroclaw was very closely related to the isolates from the other cities, and this indicated countrywide spread of the epidemic strain. The pI 5.7 beta-lactamase was produced by a single K. pneumoniae isolate for which, apart from oxyimino-beta-lactams, the MICs of beta-lactam-inhibitor combinations were also remarkably high. Sequencing revealed that this was a novel TEM beta-lactamase variant, TEM-68, specified by the following combination of mutations: Gly238Ser, Glu240Lys, Thr265Met, and Arg275Leu. The new enzyme has most probably evolved from TEM-47 by acquiring the single substitution of Arg275, which before was identified only twice in enzymes with inhibitor resistance (IR) activity. TEM-68 was shown to be a novel complex mutant TEM beta-lactamase (CMT-2) which combines strong ESBL activity with relatively weak IR activity and, when expressed in K. pneumoniae, is able to confer high-level resistance to a wide variety of beta-lactams, including inhibitor combinations. This data confirms the role of the Arg275Leu mutation in determining IR activity and documents the first isolation of K. pneumoniae producing the complex mutant enzyme.

[The use of molecular biology in the modeling of Pseudomonas aeruginosa strains recovered from nosocomial infections]. / Zastosowanie metod biologii molekularnej do typowania szczepów Pseudomonas aeruginosa izolowanych z zakazen szpitalnych.

Different methods of molecular typing (ribotyping, genomic DNA RFLP and RAPD) were tested on Pseudomonas aeruginosa strains isolated in Polish hospitals in order to elaborate a reliable typing scheme for epidemiological investigations. The combined RAPD analysis with the use of two different primers, RAPD-4 and RAPD-7, was found to have the highest discriminatory power which considering also the easiness and low time-consumption has suggested its high usefulness in studies of outbreaks caused by P. aeruginosa. Ribotyping was shown to be the least discriminatory, however, especially with the use of the PvuII restriction enzyme, this method can be very useful in revealing the genetic structure of P. aeruginosa populations persisting in hospital environments over longer periods. Clonal relations within populations of strains isolated in four different hospitals were revealed. In two of the hospitals P. aeruginosa populations demonstrated a very high diversity which suggested that infections had been caused by strains of different origins, probably introduced from other environments. P. aeruginosa strains from two remaining hospitals were found to form some clonally related clusters what revealed that in these hospitals epidemic strains of this microorganism have been circulating for prolonged periods and infecting predisposed patients.

[Susceptibility of Pseudomonas aeruginosa isolated from hospital infections to antibiotics]. / Wrazliwosc na leki szczepów pseudomonas aeruginosa izolowanych z zakazen szpitalnych.

A total of 674 clinical isolates of Pseudomonas aeruginosa were collected from 30 different hospitals located in 26 cities of Poland. These were 12 big regional hospitals, 7 large teaching hospitals, 4 specialised hospitals curing patients from the whole country, 6 small local hospitals and one regional paediatric hospital. The majority of strains were collected from patients hospitalised at ICUs (25.7%), surgical (21.7%), and internal medicine wards (9.9%). The isolates were recovered from different types of infections, mostly from respiratory tract infections (33.7%), wound infections (22.3%), and urinary tract infections (22.0%). All the isolates were subjected to antimicrobial susceptibility testing by MIC values evaluation. MICs of 13 different antibiotics (beta-lactams, aminoglycosides, chinolones) were determined by the agar dilution method. The general level of resistance of P.aeruginosa observed in the study was high, especially when compared to results of surveys obtained in other countries. Out of the antimicrobials used the highest activity in vitro was observed with meropenem, imipenem, piperacillin--tazobactam and ceftazidime. The high in vitro activity of ceftazidime was striking considering the long time of the use of this antibiotic in Polish hospitals. The highest levels of resistance were observed to some of the aminoglycosides. Populations of strains isolated in different wards or hospitals of different size were characterised by different susceptibility patterns.

How many 5S rRNA genes and pseudogenes are there in Aspergillus nidulans?

We have estimated the number of 5S rRNA genes in Aspergillus nidulans using two-dimensional agarose gel electrophoresis and hybridization to appropriate probes, representing the 5'-halves, the 3'-halves of the 5S rRNA sequence and a sequence found at the 3'-end of all known A. nidulans pseudogenes (block C). We have found 23 5S rRNA genes, 15 pseudogenes consisting of the 5'-half of the 5S rRNA sequence (of which 3 are flanked by block C) and 12 copies of block C which do not seem to be in the vicinity of 5S rRNA sequences. This number of genes is much lower than our earlier estimates, and makes our previously analyzed sample of 9 sequenced genes and 3 pseudogenes much more representative.