Multiregional dissemination of KPC-producing Klebsiella pneumoniae ST258/ST512 genotypes in Poland, 2010-14.

Objectives: In 2008-09, the KPC carbapenemase epidemiology in Poland was dominated by a Klebsiella pneumoniae ST258 KPC-2 outbreak in Warsaw and its administrative region. The aim of this study was to analyse the situation in 2010-14, with a focus on new outbreaks in other parts of the country. Methods: KPCs were detected in all suspected isolates by PCR. The detailed study was performed on 173 isolates from 2010 to 2012, and included PFGE and MLST, PCR identification of K. pneumoniae clonal group CG258 clades and potential specificity markers ( pilv-1 , IS 66 and prp ), PCR mapping of Tn 4401 transposons, and plasmid analysis by nuclease S1 profiling and PCR-based replicon typing. Results: Six hundred and eight KPC cases were identified in Poland in 2010-14, almost half of which occurred in the Warsaw region, and another half in four other areas. The new outbreaks were caused by four K. pneumoniae CG258 genotypes, different from each other and from the organisms spreading in Warsaw. The new lineages were ST258 or ST512 of clade II, and had specific compositions of potential ST258/ST512 clonal markers. The isolates produced KPC-3 encoded by Tn 4401 a or Tn 4401 b elements on plasmids with single or multiple replicons, including I2, FII K (+/-FIB K ), 'FII Y -like', X3 and R. Of other species, Citrobacter freundii ST17 and Enterobacter cloacae ST254 with KPC-2 were identified in a Warsaw hospital. Conclusions: The study showed remarkable changes in the KPC epidemiology in Poland in 2010-14, which, following the localized regional spread in the early phase, has converted into multiregional dissemination.

Molecular characteristics of KPC-producing Enterobacteriaceae at the early stage of their dissemination in Poland, 2008-2009.

After the first report in May 2008, the National Reference Center for Susceptibility Testing confirmed 113 cases of infection or colonization by KPC-producing members of the family Enterobacteriaceae in Poland by the end of 2009. The vast majority of patients were found in 18 hospitals; three patients were diagnosed at outpatient clinics. Most of the institutions were in the Warsaw area, including three hospitals with the highest numbers of cases. When available, the data on previous hospitalizations often indicated that these hospitals were the probable acquisition sites; one patient arrived from New York. The group of 119 unique isolates consisted of Klebsiella pneumoniae (n = 114), followed by Klebsiella oxytoca (n = 3), and Escherichia coli (n = 2). The K. pneumoniae isolates were dominated by the clone sequence type 258 (ST258) (n = 111); others were ST11 and ST23. The ST258 group was heterogeneous, with 28 pulsed-field gel electrophoresis (PFGE) subtypes, ∼25 plasmid profiles, and nine ß-lactamase patterns differing by KPC variants (KPC-2 mainly), and SHV-12, CTX-M-3, and TEM-1-like enzymes. Plasmids carrying bla(KPC) genes varied in size (~48 to 250 kb), structure, and conjugation potential. Transferable IncFII(K) plasmids of ~110 to 160 kb, probably pKpQIL or its derivatives, were observed in all K. pneumoniae clones and in K. oxytoca. Also prevalent were nontypeable pETKp50-like plasmids of ~50 kb, found in K. pneumoniae ST258 and E. coli isolates (ST93 and ST224). Two K. pneumoniae-E. coli pairs from single patients might represent the in vivo transfer of such plasmids. The striking diversity of KPC producers at the early stage of dissemination could result from several introductions of these bacteria into the country, their multidirectional evolution during clonal spread, and transfer of the plasmids.

blaCTX-M genes in escherichia coli strains from Croatian Hospitals are located in new (blaCTX-M-3a) and widely spread (blaCTX-M-3a and blaCTX-M-15) genetic structures.

CTX-M-producing Escherichia coli isolates from three Croatian hospitals were analyzed. All bla(CTX-M-15) genes and one bla(CTX-M-3a) gene resided in widely spread ISEcp1 transposition modules, but other bla(CTX-M-3a) genes were in a new configuration with two IS26 copies, indicating a new event of gene mobilization from a Kluyvera ascorbata genome. The study confirmed the role of the E. coli ST131 clonal group with IncFII-type plasmids in the spread of bla(CTX-M-15) and of IncL/M pCTX-M3-type plasmids in the dissemination of bla(CTX-M-3a).

Molecular survey of beta-lactamases conferring resistance to newer beta-lactams in Enterobacteriaceae isolates from Polish hospitals.

The first national survey of resistance to newer beta-lactams in nosocomial populations of Enterobacteriaceae in Poland was performed. The study covered all nonrepetitive enterobacterial isolates cultured from specimens from inpatients in 13 regional secondary-care hospitals from November 2003 to January 2004. Among 2,388 isolates, the predominant species was Escherichia coli (59.6%), followed by Proteus mirabilis (14.5%) and Klebsiella spp. (8.5%). The frequency of extended-spectrum beta-lactamases (ESBLs) was very high, with ESBLs present in 11.1% of all isolates and 40.4% of Klebsiella pneumoniae isolates, the latter value greatly exceeding that for E. coli (2.5%). The contribution of outbreak isolates was significant, resulting, for example, in a particularly high rate of ESBL producers among Serratia marcescens isolates (70.8%). The pool of ESBL types was overwhelmingly dominated (81.7%) by CTX-M-like beta-lactamases CTX-M-3 (80.6%) and CTX-M-15, with SHV types (17.5%; SHV-2, SHV-5, and SHV-12) and sporadic TEM-like enzymes (0.7%; TEM-19 and TEM-48) being the next most frequent. Acquired AmpC-type cephalosporinases were observed exclusively in P. mirabilis, in 20.5% of the isolates of this species (compared with the frequency of ESBL producers of 11.5% of P. mirabilis isolates). All these cephalosporinases (CMY-12, CMY-15, and a novel variant, CMY-38) originated from Citrobacter freundii. Four isolates of E. coli (two isolates), K. pneumoniae (one isolate), and P. mirabilis (one isolate) produced class A inhibitor-resistant beta-lactamases (TEM-30, TEM-32, TEM-37, and SHV-49), being the first of such producers identified in Poland. The survey documented both specific and more global characteristics of the epidemiology of the beta-lactamase-mediated resistance in enterobacteria from Polish hospitals and demonstrated that the ESBL frequency has reached an alarming level.

Highly variable penicillin resistance determinants PBP 2x, PBP 2b, and PBP 1a in isolates of two Streptococcus pneumoniae clonal groups, Poland 23F-16 and Poland 6B-20.

Penicillin-binding proteins (PBPs) in representatives of two Streptococcus pneumoniae clonal groups that are prevalent in Poland, Poland 23F-16 and Poland 6B-20, were investigated by PBP profile analysis, antibody reactivity pattern analysis, and DNA sequence analysis of the transpeptidase (TP) domain-encoding regions of the pbp2x, pbp2b, and pbp1a genes. The isolates differed in their MICs of beta-lactam antibiotics. The majority of the 6B isolates were intermediately susceptible to penicillin (penicillin MICs, 0.12 to 0.5 microg/ml), whereas all 23F isolates were penicillin resistant (MICs, >or=2 microg/ml). The 6B isolates investigated had the same sequence type (ST), determined by multilocus sequence typing, as the Poland 6B-20 reference strain (ST315), but in the 23F group, isolates with three distinct single-locus variants (SLVs) in the ddl gene (ST173, ST272, and ST1506) were included. None of the isolates showed an identical PBP profile after labeling with Bocillin FL and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and only one pair of 6B isolates and one pair of 23F isolates (ST173 and ST272) each contained an identical combination of PBP 2x, PBP 2b, and PBP 1a TP domains. Some 23F isolates contained PBP 3 with an apparently higher electrophoretic mobility, and this feature also did not correlate with their STs. The data document a highly variable pool of PBP genes as a result of multiple gene transfer and recombination events within and between different clonal groups.

Clonal diversity and resistance mechanisms in tetracycline-nonsusceptible Streptococcus pneumoniae isolates in Poland.

The frequency of tetracycline resistance in Streptococcus pneumoniae isolates in Poland is one of the highest in Europe. The aim of this study was to analyze the clonal diversity and resistance determinants of tetracycline-nonsusceptible S. pneumoniae isolates identified in Poland and to investigate the effect of tetracycline resistance on their susceptibilities to tigecycline, doxycycline, and minocycline. We have analyzed 866 pneumococcal isolates collected from 1998 to 2003 from patients with respiratory tract diseases, and 242 of these (27.9%) were found to be resistant to tetracycline. All of the resistant isolates were characterized by testing of their susceptibilities to other antimicrobials, serotyping, pulsed-field gel electrophoresis (PFGE), and identification of tetracycline resistance genes and transposons. Selected isolates representing the main PFGE types were analyzed by multilocus sequence typing. Among the isolates investigated, 27 serotypes and 146 various PFGE patterns, grouped into 90 types, were discerned. The most common PFGE type, corresponding to serotype 19F and sequence type 423, was represented by 22.3% of all of the tetracycline-resistant isolates. The tet(M) gene was the sole resistance gene in the group of isolates studied, and in over 96% of the isolates, the Tn916 family of tet(M)-containing conjugative transposons was detected. Several isolates contained specific variants of the transposons, the Tn1545-like, Tn3872-like, or Tn2009-like element. The correlation between the MICs of tetracycline, doxycycline, and minocycline was revealed, whereas no cross-resistance to tetracycline and tigecycline was observed.

Molecular epidemiology of acquired-metallo-beta-lactamase-producing bacteria in Poland.

We have analyzed 40 metallo-beta-lactamase (MBL)-producing isolates of Pseudomonas aeruginosa (n = 38), Pseudomonas putida (n = 1), and Acinetobacter genospecies 3 (n = 1) from 17 hospitals in 12 cities in Poland that were identified in 2000 to 2004. Pulsed field gel electrophoresis typing classified the P. aeruginosa isolates into eight types, with two types differentiated further into subtypes. Each of the types was specific either to a given center or to several hospitals of the same or neighboring geographic area. Almost all of the organisms produced beta-lactamase VIM-2; the only exceptions were several P. aeruginosa isolates from two centers which expressed VIM-4. The bla(VIM) genes resided exclusively within class 1 integrons, and these were located in either chromosomal or plasmid DNA. PCR-restriction fragment length polymorphism study of the variable regions of the integrons, followed by DNA sequencing, revealed the presence of eight different, mostly novel gene cassette arrays, six of which contained bla(VIM-2) and two of which contained bla(VIM-4). The occurrence of the integron variants correlated well with the geographic distribution of the MBL-producing organisms, and this suggested that their emergence in particular parts of the country had been likely due to a number of independent events. The following regional dissemination of MBL producers could be attributed to various phenomena, including their clonal spread, horizontal transmission of resistance determinants, or both. All of the data collected in this study revealed that even at this early stage of detection, the epidemiological situation concerning MBL producers in Poland has already been complex and very dynamic.

Multilocus sequence types, serotypes, and variants of the surface antigen PspA in Streptococcus pneumoniae isolates from meningitis patients in Poland.

Meningitis caused by Streptococcus pneumoniae represents an important factor of morbidity and mortality in humans. In a significant number of cases, this disease is associated with specific clones of the organism, the so-called invasive pneumococcal clones. The aim of the study was to analyze 156 S. pneumoniae isolates identified as etiological agents of meningitis in Poland in the years 1997 through 2002. The isolates were characterized by multilocus sequence typing (MLST), and the results were compared with those obtained by pulsed-field gel electrophoresis (PFGE) and with the MLST data on invasive pneumococci from other countries. Eighty-nine different sequence types were found in the group of isolates, 50 of which had been known before including 19 of the major invasive clones. However, a significant fraction of the isolates possessed novel combinations of known and new MLST alleles. The majority of penicillin-nonsusceptible isolates belonged to the group of international multiresistant clones (Spain(23F)-1, Spain(6B)-2, Spain(9V)-3, Poland(23F)-16, and Poland(6B)-20), which underlined the importance of these in the dissemination of antimicrobial resistance. The results of the MLST analysis correlated well with the PFGE data, thus again demonstrating good congruence between the two typing methods for S. pneumoniae. The majority of the isolates (95.5%) belonged to families 1 or 2 of the surface protein PspA, confirming its potential usefulness as the vaccine antigen candidate.

Evolution of TEM-type extended-spectrum beta-lactamases in clinical Enterobacteriaceae strains in Poland.

Seventeen extended-spectrum beta-lactamase (ESBL)-producing isolates of the family Enterobacteriaceae recovered from 1998 to 2000 in hospitals of five different cities in Poland were analyzed. They expressed several TEM-type ESBLs, TEM-4, TEM-29, TEM-85, TEM-86, TEM-93, and TEM-94. TEM-85 (L21F, R164S, E240K, T265M), TEM-86 (L21F, R164S, A237T, E240K, T265M), TEM-93 (M182T, G238S, E240K), and TEM-94 (L21F, E104K, M182T, G238S, T265M) were identified for the first time. Including the enzymes described earlier, TEM-47, TEM-48, TEM-49, and TEM-68, the group of known ESBLs of the TEM family produced by enterobacteria in Polish hospitals has increased to 10 variants. Comparative sequence analysis of the genes coding for all these beta-lactamases revealed a view of their possible evolution, which, apart from the gradual acquisition of various mutations, could also have involved recombination events. Two different bla(TEM-1) gene alleles were precursors of the ESBL genes: bla(TEM-1A), which was the ancestor of bla(TEM-93), and bla(TEM-1F), from which all the remaining genes originated. The evolution of the bla(TEM-1F)-related genes most probably consisted of three major separate lineages, one of which, including bla(TEM-4), bla(TEM-47), bla(TEM-48), bla(TEM-49), bla(TEM-68), and bla(TEM-94), was highly structured itself and could have been initiated by the bla(TEM-25) gene, identified exclusively in France so far. Plasmid fingerprinting analysis revealed a high degree of diversity of plasmids carrying related bla(TEM) genes, which suggested either the intense diversification or transposition of bla(TEM) genes between different plasmids or some contribution of convergent evolution. The results of this study clearly demonstrate that the environment of Polish hospitals has been highly favorable for the rapid evolution of ESBLs.

A new method of DNA preparation for pulsed-field gel electrophoresis analysis.

We have developed a new, labour-saving method of preparation, handling and treatment of DNA-containing agarose plugs for pulsed-field gel electrophoresis (PFGE). A plastic mould in which plugs are formed and supported during DNA purification and digestion was designed and successfully tested in a prototype device.

Characteristics of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis isolated from the nasopharynges of asymptomatic children and molecular analysis of S. pneumoniae and H. influenzae strain replacement in the nasopharynx.

Nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in 226 children in different settings (in a crèche [day care center], in an orphanage, and at home) during two seasons (winter and spring) was studied. The rates of carriage of S. pneumoniae and H. influenzae were markedly higher in the crèche and in the orphanage than in the home setting (e.g., 56.5, 63.3, and 25.9%, respectively, for S. pneumoniae in winter). Approximately 80% of the S. pneumoniae isolates identified in the crèche and in the orphanage belonged to the serotypes represented in the seven-valent pneumococcal vaccine, and 4.4% of the children were colonized by H. influenzae type b. Almost all H. influenzae isolates were fully susceptible to the antimicrobial agents tested, and only five (3.6%) produced beta-lactamase; in contrast, 100% of the M. catarrhalis isolates were beta-lactamase positive. Among S. pneumoniae isolates, 36.2% were nonsusceptible to penicillin (PNSP) and 11.8% were fully resistant to penicillin (PRP). All PNSP isolates were obtained from children at the crèche and at the orphanage but not among children brought up at home, and all PRP isolates showed a multiresistant phenotype. Colonization by PRP isolates correlated well with prior treatment with beta-lactams. For the majority of children colonized at both sampling times, strain replacement of S. pneumoniae and H. influenzae was observed; long-term colonization by a single strain was rare.

[Chlamydia pneumoniae infection in patients with advanced carotid atherosclerosis]. / Zakazenia Chlamydia pneumoniae u chorych z zaawansowana miadyca tetnic szyjnych.

Despite the growing number of scientific reports showing different markers of infection caused by Chlamydia pneumoniae in patients with advanced atherosclerosis, there is still no clear confirmation of a pathogenetic link between this infection and atherogenesis. The aim of the present study was to investigate the presence C. pneumoniae DNA in peripheral blood mononuclear cells and carotid endarterectomy samples obtained from patients with advanced atherosclerosis according to the presence specific antibodies against C. pneumoniae in serum. The levels of IgG, IgM and IgA antibodies to C. pneumoniae were determined by enzyme immunoassay (EIA) in sera of 36 patients with advanced atherosclerosis undergoing carotid endarterectomy. The presence of C. pneumoniae DNA in the carotid samples and in peripheral blood mononuclear cells was investigated by a nested polymerase chain reaction (PCR). We have not confirm the presence of C. pneumoniae DNA either in the carotid endarterectomy samples or in peripheral blood mononuclear cells, both in patients having the specific antibodies against C. pneumoniae and in seronegative patients. In the studied group of patients with advanced carotid atherosclerosis there was no correlation between the presence of specific antibodies against C. pneumoniae and the presence of C. pneumoniae DNA in endarterectomy samples and peripheral blood mononuclear cells.

The new strategy for allele identification of the genes coding for pertussis toxin subunit S1 (ptx S1) and pertactin (prn) in Bordetella pertussis.

Bordetella pertussis strains demonstrate polymorphism in toxin subunit S1 (PT S1) and pertactin (Prn), which belong to major protective antigens of the pathogen. Changes in the distribution of particular alleles of ptxS1 and prn genes in local B. pertussis populations have been proposed as possible factors influencing the vaccination effectiveness. We have developed a new methodology for the identification of the alleles, which eliminates the necessity of DNA sequencing. The approach is based on the evaluation of the number of sequence repeats and detection of specific nucleotides at polymorphic sites of the genes, and utilizes products of their full or partial PCR amplification. The approach is available for a laboratory with standard equipment. The total conformity of the strategy with the DNA sequencing-based approach was proved on the full set of reference strains and a group of Polish clinical isolates. The new methodology was used to investigate a collection of 120 Polish B. pertussis strains isolated from the 1960s to 2001. Similarly to findings from other countries and to earlier Polish data, the tendency to change the vaccine types of PT S1 and Prn by the antigenically different ones was observed.

Improvement in the laboratory recognition of lyme borreliosis with the combination of culture and PCR methods.

BACKGROUND: Lyme disease is a multisystem, multistage infection caused by three genospecies of the Borrelia burgdorferi sensu lato species. The diagnosis of Lyme disease is based on a history of tick-bite, physical examination, and serological tests. In the seronegative patients with Lyme borreliosis symptoms, additional testing should be introduced. METHODS: The study group was composed of 240 hospitalized patients presented with various clinical symptoms suggesting Lyme borreliosis: 221 of the patients with neurological abnormalities and 19 with oligoarticular arthritis. Citrated blood and serum samples were collected from the patients for culture and serological examination, respectively. Moreover, 173 cerebrospinal and 6 synovial fluid samples were tested. New oligonucleotide primers based on B. burgdorferi sensu lato 16SrRNA gene sequences were designed for the detection of the bacteria in blood, cerebrospinal, and synovial fluid specimens with PCR. Levels of specific antibodies were measured in serum, cerebrospinal fluid and synovial fluid samples using ELISA and Western blot. B. burgdorferi spirochetes from blood, cerebrospinal fluid, and synovial fluid samples were cultured in cell line. Extracted and purified B. burgdorferi DNA was identified by PCR with new oligonucleotide primers. Then three genospecies were identified by PCR amplification with other primer sets specific for 16S rDNA and/or by the restriction fragment length polymorphism of 23S(rrl)-5S(rrf). RESULTS: Bacterial DNA were found in samples from 32 patients, including 28 patients with neuroborreliosis and 4 with Lyme arthritis. B. burgdorferi-specific IgM and/or IgG serum antibodies were detected in 14 of these patients. Fourteen strains of Borrelia garini, 4 strains of Borrelia afzelii and 1 strain of B. burgdorferi sensu stricto were identified by PCR. Genospecies were not recognized in 13 specimens. CONCLUSIONS: The procedure can be a rapid and sensitive diagnostic method for the detection of etiological agents in clinical materials derived from patients with the clinical symptoms of Lyme borreliosis. It can be utilized for both basic research as well as routine laboratory diagnosis.

Ceftazidime-hydrolysing CTX-M-15 extended-spectrum beta-lactamase (ESBL) in Poland.

The CTX-M-15 extended-spectrum beta-lactamase (ESBL) was recently identified in Enterobacteriaceae isolates in India, and demonstrated significant hydrolytic activity against ceftazidime, in contrast to the majority of CTX-M enzymes. CTX-M-15 differs from CTX-M-3, which is one of the most prevalent ESBLs in Poland, by only a single amino acid change (Asp-240-->Gly). Three cefotaxime- and ceftazidime-resistant Enterobacteriaceae isolates, recovered during 1998-2000 in two Polish hospitals, were found to produce CTX-M-15. Similar to those from India, the isolates contained the ISEcp1 insertion sequence located upstream of the bla(CTX-M-15) gene, which has been recently demonstrated to mobilize 3'-adjacent genes to transfer between DNA replicons. However, its different position with respect to the beta-lactamase gene indicated the independent selection of the ESBL gene in the two countries.

Intercenter reproducibility of binary typing for Staphylococcus aureus.

The reproducibility of the binary typing (BT) protocol developed for epidemiological typing of Staphylococcus aureus was analyzed in a biphasic multicenter study. In a Dutch multicenter pilot study, 10 genetically unique isolates of methicillin-resistant S. aureus (MRSA) were characterized by the BT assay as presented by van Leeuwen et al. [J. Clin. Microbiol. 2001 39 (1) 328]. The BT assay, including a standardized DNA extraction protocol was performed in duplicate in eleven medical microbiology laboratories. Two different hybridization detection procedures were applied and a prelabeled DNA sample as process control was included. Only three laboratories accurately identified all strains. Divergence in technical procedures resulted in misinterpretation due to an increasing number of faint or absent hybridization signals in combination with high background staining. The binary type of the process control was determined correctly by all participating laboratories. The feasibility of the BT protocol was related directly to the skill of the laboratory personnel. On the basis of the national study, we concluded that the DNA extraction protocol needed modification to improve DNA yield and purity. Subsequently, seven European laboratories participated in an international study to determine the reproducibility of the modified BT protocol. Each center was asked to analyze 10 DNA samples previously extracted from 10 MRSA strains (phase 1) and, additionally, to analyze 10 MRSA strains, using the standardized or their in-house DNA isolation protocol (phase 2). A prelabeled DNA process control sample was included again. The binary types of all DNA samples were identified correctly by all but one laboratories. This latter laboratory diverged from the protocol by adding an excess of labeled DNA to the hybridization mixture, resulting in a high background and, therefore, noninterpretable BT results. All centers produced identical BT results for the process control. Five of the seven centers correctly identified the binary types of all 10 MRSA strains in phase 2 of the international study. Three of these centers used their in-house DNA extraction protocol. Divergence from the standard BT protocol in the remaining two centers resulted in no interpretable BT data for the 10 MRSA strains. The study demonstrated that each center that followed the BT protocol to the letter could generate reproducible results, irrespective whether or not an in-house DNA isolation protocol was used. The current BT protocol thus represents a simple method generating robust, reproducible genotype data for S. aureus strains.

Countrywide spread of CTX-M-3 extended-spectrum beta-lactamase-producing microorganisms of the family Enterobacteriaceae in Poland.

Eighty-four clinical isolates of the family Enterobacteriaceae, recovered from 1998 to 2000 in 15 hospitals in 10 Polish cities, were analyzed. All the isolates produced beta-lactamases with pIs of 8.4 and 5.4, and the pI 8.4 enzymes were demonstrated to hydrolyze cefotaxime but not ceftazidime in the in vitro bioassay. PCR analysis and DNA sequencing have revealed that in all cases the pI 8.4 beta-lactamase was probably the CTX-M-3 extended-spectrum beta-lactamase (ESBL) variant, which was originally identified in 1996 in Praski Hospital in Warsaw. In the majority of isolates, bla(CTX-M-3) genes resided within large conjugative plasmids with similar fingerprints, which, in the context of the high degree of diversity of the randomly amplified polymorphic DNA types of the isolates, suggested that horizontal transfer of plasmids was likely the main mechanism of CTX-M-3 spread. The dissemination of plasmids was probably preceded by the center-to-center transmission of several strains, as indicated by the identification by pulsed-field gel electrophoresis of closely related or possibly related Klebsiella pneumoniae, Escherichia coli, and Citrobacter freundii isolates in five different hospitals. CTX-M-3-producing organisms revealed a very high degree of diversity in beta-lactam resistance levels and patterns. This was attributed to several factors, such as the production of other beta-lactamases including additional ESBLs, possible quantitative variations in CTX-M-3 expression, segregation of AmpC derepressed mutants, and permeability alterations.