Molecular Epidemiology and Antimicrobial Resistance of ESBL- and Carbapenemase-Producing Uropathogenic E. coli and K. pneumoniae in Community-Acquired UTIs in Baghdad, Iraq

3.4 β-lactamase inhibitory activity

Inhibitor assays were conducted with 10 μL of boronic acid or cloxacillin added to antibiotic discs to detect group A carbapenemases and AmpC β-lactamases. A positive result was defined as an increase in the inhibition zone of more than 5 mm. Seven E. coli isolates were initially included in the study; however, two were excluded due to the absence of cephalosporin resistance. Four isolates (1, 2, 3, and 49) exhibited resistance to all tested antibiotics, including cephalosporins and carbapenems. However, isolate 37 was sensitive to carbapenems but resistant to CTX and CAZ, indicating the development of an ESBL of the CTX-M type.

3.5 Carbapenemase and ESBL confirmation

Boronic acid significantly increased inhibition zones for MEM, ETP, and DOR in isolates 1, 2, 3, and 49, confirming K. pneumoniae carbapenemase (KPC) production. However, cloxacillin and imipenem + EDTA (IPM/IPI) did not affect inhibition zones, ruling out AmpC β-lactamases and metallo-β-lactamases. MIC was determined using Etest strips. For isolate 37, CTX, CTX/CTXL, CAZ, CAZ/CAZL, CFEP, and CFEP/FEPL were tested. For isolates 1, 2, 3, and 49, imipenem (IPM), meropenem (MEM), and imipenem + EDTA (IPM/IPI) were used. MIC values ranged from 8 to 24 mg/L for IPM and MEM (Figure 2), confirming carbapenemase production. There were no significant changes in susceptibility with the use of IPM/IPI strips, effectively ruling out metallo-β-lactamase production. Isolate 37 exhibited minimum inhibitory concentrations (MICs) greater than 256 mg/L for cefotaxime (CFX), cefazolin (CFZ), and cefepime (CEFP), supporting the classification of this isolate as an extended-spectrum β-lactamase (ESBL) producer. The use of CFX/CFXL, CFZ/CFZL, and CEFP/CEFPL combination strips enables the identification of ESBL-producing isolates by comparing results against established reference thresholds: CFX MICs ≥ 0.5 mg/L with a CFX/CFXL ratio ≥ 8; CFZ MICs ≥ 1 mg/L with a CFZ/CFZL ratio ≥ 8; or CEFP MICs ≥ 0.25 mg/L with a CEFP/CEFPL ratio ≥ 8. In the case of isolate 37, the results obtained with the CFZ/CFZL and CEFP/CEFPL strips met these criteria, thereby confirming the presence of ESBL activity.

image011.jpg

Figure 2. Electrophoretic profiles generated by M13 and BOX fingerprinting molecular typing methods using 2% agarose gel

A: Profile of E. coli isolates; B: Profile of K. pneumoniae isolates; M: GeneRuler^TM 1kb DNA ladder molecular weight marker.

Tests were conducted on K. pneumoniae isolates using inhibitors to assess β-lactamase activity. Cloxacillin was added to discs containing ceftazidime (CAZ), amoxicillin/clavulanic acid (AMX/CA), cefotaxime (CTX), cefepime (CFEP), and imipenem (IPM). The lack of alterations in inhibitory zones suggested that AmpC β-lactamases were not produced by these isolates. Another inhibitor, boronic acid, was tested with imipenem (IPM), meropenem (MEM), doripenem (DOR), and ertapenem (ETP). No significant changes were observed in inhibition zones, except in a group of seven isolates where an increased inhibition zone in the DOR + boronic acid disc suggested the presence of a carbapenemase.

The Etest technique was used to verify the existence of ESBLs. With ranges of 8 mg/L to >256 mg/L for cefotaxime (CTX), 12 mg/L to >256 mg/L for ceftazidime (CAZ), and 24 mg/L to >256 mg/L for CFEP, the minimum inhibitory concentrations (MIC) for all three medications were noticeably high. Resistance to these medicines was validated by these high MIC values. Furthermore, when clavulanic acid was diffused into CTXL, CAZL, or FEPL zones, the inhibition ellipse on CTX, CAZ, or CFEP strips showed a "ghost zone" or distortion, which suggested the existence of ESBLs. This trait was most noticeable in K. pneumoniae isolate 24, which was sensitive to carbapenems but resistant to cephalosporins. The molecular typing techniques analyzed the genetic relationships between isolates, including M13-PCR, BOX-PCR fingerprinting, and MLST. In E. coli isolates, M13-PCR fingerprinting established a genetic link between isolates 1, 2, and 49, while BOX-PCR fingerprinting demonstrated similarities between isolate 3 and the other E. coli isolates. These analyses confirmed a close genetic relationship among isolates 1, 2, 3, 49, and 37 from Yarmouk Teaching Hospital. Notably, isolates 1 and 2 were collected from the same patient at different times, one from the nephrology unit and another three months later from the emergency room, while isolates 3, 49, and 37 were obtained from different biological samples of unrelated patients across multiple hospital units.

Among the 19 selected K. pneumoniae isolates, two distinct genetic profiles, B and C, were identified. Profile B included 13 isolates from different patients across three hospitals. In contrast, Profile C consisted of isolates collected from a single patient within a four-day interval at two hospitals, namely Al-Shaab General Hospital and Baghdad Medical City. In contrast, molecular typing of Enterobacter isolates and the seven selected K. pneumoniae isolates did not provide sufficient resolution to establish a clear genetic relationship. Further details on sample origins, hospital sources, and electrophoretic profiles are provided in Table 3. The MLST technique was performed to characterize the bacterial isolates further and confirm their clonal relationships. This method involves amplifying internal fragments of seven housekeeping genes, which are constitutive and species-specific [31]. Based on nucleotide variations, each isolate was assigned an allelic profile that, when analyzed against a reference database, allows for identifying different sequence types (STs) through homology with previously described profiles. Klebsiella pneumoniae isolates 35 and 40 (Profile B), isolate 24 (Profile C), and E. coli isolates 1, 3, 49, and 37 (Profile A) were all subjected to MLST. Additionally, it was performed on K. pneumoniae isolates 7, 17, and 29, which exhibited resistance to cephalosporins and intermediate resistance to carbapenems in antibiograms. In E. coli isolates 1, 3, 49, and 37, the globally disseminated clone ST131 [32] was identified, which is recognized for its role in the spread of the CTX-M-15 enzyme. The allelic profile of these isolates was determined by sequencing seven housekeeping genes [31]: adk, fumC, gyrB, icd, mdh, purA, and recA. The detailed allelic profiles of these isolates are provided in Table 4.

Table 3. Strain identification, sample type, hospital service, unit of origin, and electrophoretic profiles of the studied isolates

AY-TH: Al-Yarmouk Teaching Hospital; ASGH: Al-Shaab General Hospital; BMC: Baghdad Medical City; A: found in E. coli isolates; B: found in K. pneumoniae isolates with one grouping, and X: found in K. pneumoniae isolates with a distinct genetic profile.

Table 4. Allelic profile of E. coli isolates

P: Present (indicates detecting the characteristic nucleotide sequence for each housekeeping gene in ST131 isolates, and ST131 is a globally disseminated clone frequently associated with the spread of the CTX-M-15 extended-spectrum β-lactamase enzyme).

To properly describe the bacterial isolates and validate their clonal connection, the MLST methodology was applied to the most representative profiles that were generated using the earlier techniques. Following analysis of the profiles in a database, the various sequence types may be identified based on their similarity with the previously mentioned profiles. This technique was used for E. coli isolates 1, 3, 49, and 37 (Profile A) and for K. pneumoniae isolates 35 and 40 (Profile B) and 24 (Profile C). It was also applied to K. pneumoniae isolates 7, 17, and 29, showing a resistance profile to cephalosporins and intermediate resistance to carbapenems in the antibiograms. In E. coli isolates 1, 3, 49, and 37, the globally disseminated clone ST131 was identified, which is known for its role in spreading the CTX-M-15 enzyme (Table 4). The allelic profile of these isolates was determined by analyzing the nucleotide sequences of seven housekeeping genes, including adk, fumC, gyrB, icd, mdh, purA, and recA. The detailed allelic profiles of these isolates are presented in Table 4.

ST15, a clone that has recently been linked to the spread of CTX-M enzymes in isolates of K. pneumoniae, was found in isolates 35, 40, 7, 17, and 29 [24]. A distinct ST was found in isolate 24, which is in line with the profiles derived using the BOX fingerprinting typing technique. The closest ST to the one discovered in this isolate is ST20, whose nucleotide sequences of the genes under study vary from isolate 24 only in the mdh gene, as shown in Table 5. The ST detected in this isolate is not mentioned in the database that was utilized. Considering the nucleotide sequences of the housekeeping genes gapA, infB, mdh, pgi, phoE, rpoB, and tonB, Table 5 shows the allelic profile for K. pneumoniae isolates 35, 40, 24, 7, 17, and 29 [33].

Table 5. Allelic profile of K. pneumoniae isolates

“I”: identical allelic sequences matching those found in ST15 or the nearest sequence type; Variant: a sequence difference, as seen in the mdh gene of isolate 24; Profile in Database: reflects whether the full allelic profile corresponds to an existing entry in the MLST database.

3.6 Genetic determinants of resistance

PCR analysis was performed to detect resistance genes (Figure 3), including the carbapenemases bla_KPC, bla_OXA-48, and bla_OXA-58, and ESBLs, bla_CTX, bla_TEM, bla_SHV, bla_DHA, bla_CMY, and bla_CEFT. Enterobacter isolates 28, 29, 30, and 32 tested negative for bla_CTX and bla_KPC and were excluded from further study. Their resistance may be due to alternative mechanisms, such as porin modifications or efflux pump activity. In E. coli isolates 1, 2, 3, and 49, bla_KPC amplification correlated with carbapenem resistance and increased inhibition zones in boronic acid disk tests. Sequencing confirmed the bla_KPC-2 variant, the most globally widespread KPC enzyme, primarily found in K. pneumoniae. The bla_TEM gene was also detected, identifying the TEM-1 β-lactamase [34]. No amplification was observed for bla_CTX, bla_OXA-48, bla_OXA-58, or bla_SHV. The E. coli ST131 clone, known for spreading the CTX-M-15 enzyme, was first reported carrying bla_KPC-2 in Ireland [35]. Its presence in this highly transmissible lineage raises concerns about the rapid dissemination of carbapenem resistance.

image013.jpg

Figure 3. PCR products related to amplifying the bla_KPC gene of 871 bp (base pairs) in a 1% agarose gel

M: GeneRuler^TM 1kb DNA ladder molecular weight marker.

PCR amplification detected the bla_CTX gene in E. coli isolate 37 and K. pneumoniae isolates 25, 26, 28, 30, 31, 36, 39, 40, 35, 24, 38, 41, 42, and 48, all of which were resistant to CTX and CEFZ but susceptible to carbapenems. Sequencing confirmed the bla_CTX-M-15 variant in isolates 37, 36, 40, 35, and 24, a globally prevalent enzyme conferring resistance to third- and fourth-generation cephalosporins, such as CTX, CEFZ, and cefepime (CFEP). The bla_TEM gene was also identified, and sequencing of E. coli isolate 37 and K. pneumoniae isolates 40 and 24 confirmed the bla_TEM-1 variant, responsible for resistance to first-generation penicillins and cephalosporins. The bla_SHV gene was absent in E. coli isolate 37 but detected in K. pneumoniae isolates 35, 40, and 24. Sequencing revealed bla_SHV-28 in isolates 35 and 40 and bla_SHV-12 in isolate 24. The SHV-12 enzyme demonstrated enhanced hydrolytic activity against cephalosporins, particularly CEFZ and CTX, while SHV-28 preferentially hydrolyzes CTX over CEFZ, reflecting the resistance patterns observed in isolates 35 and 40. The bla_OXA-48 gene was absent in all E. coli and K. pneumoniae isolates. Among seven K. pneumoniae isolates (7, 17, 29, 43, 33, 46, and 47) with susceptibility or intermediate resistance to IPM and MEM but resistance to doripenem (DOR) and ertapenem (ETP), no amplification of bla_KPC, bla_OXA-48, bla_DHA, bla_CMY, or bla_CEFT was observed. However, isolates 7, 17, 29, and 43, resistant to CTX and CEFZ, tested positive for bla_CTX, bla_TEM, and bla_SHV (Figure 4). Sequencing of isolate 17 confirmed bla_CTX-M-15, bla_TEM-1, and bla_SHV-1. In isolates 33, 46, and 47, which showed intermediate or susceptible levels to CTX but resistance to ceftazidime (CEFT) and CFEP, only the bla_SHV-1 gene was identified, conferring resistance to first-generation penicillins and cephalosporins.

image015.jpg

Figure 4. PCR products related to amplifying the 544 bp bla_CTX gene in a 1% agarose gel

M: GeneRuler^TM 1kb DNA ladder molecular weight marker.

Table 6 outlines β-lactamase genes, porin presence, electrophoretic profiles, and sequence types of E. coli and K. pneumoniae isolates. Most E. coli belonged to ST131 harboring bla_KPC-2, while K. pneumoniae mainly carried bla_CTX-M-15 and belonged to ST15, indicating clonal and gene-specific resistance patterns.

Resistance to β-lactam antibiotics can result from altered outer membrane permeability due to reduced expression or absence of porin-encoding genes. Since the seven K. pneumoniae isolates (7, 17, 29, 33, 43, 46, and 47) with susceptibility or intermediate resistance to IPM and MEM but resistance to DOR and ETP tested negative for bla_KPC, the presence of the OmpK35 and OmpK36 porins was investigated. Most non-ESBL-producing K. pneumoniae express both porins, while ESBL-producing isolates commonly express only OmpK36. The absence of both porins has been linked to β-lactam resistance. Recent studies suggest that OmpK35 is associated with ceftazidime susceptibility, whereas OmpK36 expression correlates with cefotaxime susceptibility. Loss of OmpK36 is linked to increased resistance to cefoxitin and carbapenems in β-lactamase-producing strains. The OmpK35 gene was found in all isolates, while OmpK36 was detected in only five (7, 17, 29, 43, and 46). OmpK36 was absent in isolates 33 and 47, which might account for their increased vulnerability to ceftazidime. The absence of OmpK36 may contribute to increased carbapenem resistance. Further analysis is needed to determine porin expression levels [36], as the presence of a gene does not guarantee its expression. Based on these findings, the following isolates were selected for further study: E. coli ST131 isolates 1, 2, 3, and 49 producing KPC-2 carbapenemase; E. coli ST131 isolate 37 producing ESBL CTX-M-15 (Profile A); K. pneumoniae isolate 24 with a distinct sequence type (Profile C); K. pneumoniae isolates 35 and 40 with ST15 (Profile B); and K. pneumoniae isolates 7, 17, 29, and 43 with distinct electrophoretic profiles (X) but sharing ST15, all producing ESBL_CTX-M-15.

Table 6. Primary resistance genes identified in the studied isolates and their corresponding hospitals of origin

NA: Not applicable or not available; ST: Sequence type based on molecular typing; EP: Electrophoretic profile (distinct molecular profiles of isolates).

Table 7. Primers designed in the laboratory and PCR conditions used to determine the genetic environment of the bla_KPC-2 gene

ISKpn8: internal region of the ISKpn8 insertion sequence; ISKpn8T: region between ISKpn8 and the truncated bla_TEM gene; KPCT: region between the truncated bla_TEM gene and the bla_KPC-2 gene; F: forward primer; A: reverse primer.

3.7 Dissemination of genetic determinants of resistance

The spread of ESBLs in Enterobacteriaceae is driven by clonal dissemination and mobile genetic elements like plasmids, transposons, and integrons. The bla_KPC-2 gene is carried by the Tn3-type transposon Tn4401, which includes tnpA, tnpR, and insertion sequences ISKpn6 and ISKpn7. Tn4401, found in various species and regions, exists in three isoforms (a, b, and c) that differ by 100–200 bp deletions upstream of bla_KPC-2, highlighting its high mobility and role in resistance gene spread. Other variants of Tn4401 have been described, mainly involving modifications in the upstream region of bla_KPC-2, indicating its instability. In China, a novel genetic environment for bla_KPC-2 was identified, featuring an ISKpn6-like sequence downstream of the gene, a newly described ISKpn8 insertion sequence upstream, and a truncated bla_TEM gene [37, 38]. For E. coli isolates 1, 2, 3, and 49, which carry bla_KPC-2, the presence of Tn4401 elements was investigated using PCR with primers targeting ISKpn7, ISKpn6, tnpA, and tnpR. Only ISKpn6 was amplified, and sequencing confirmed its identity with the previously described Tn4401 transposon. No amplification of tnpA, tnpR, or ISKpn7 was observed. Given this, primers were designed to detect the novel genetic structure described in China, targeting ISKpn8, the region between ISKpn8 and the truncated bla_TEM gene, and the region between bla_TEM and bla_KPC-2, as detailed in Table 7 [37].

Genes upstream of bla_KPC-2 could not be identified since no amplification was observed in any of the regions examined. The genetic environment of bla_CTX-M-15 was also examined, given its association with mobile elements like the ISEcp1 insertion sequence, which plays a key role in mobilizing bla_CTX-M genes. Additional elements, such as IS26 and IS903, also contribute to gene transfer. In E. coli isolates 37 and K. pneumoniae 24, 35, 40, 7, 17, 29, and 43, ISEcp1 was consistently identified upstream of bla_CTX-M-15. With the exception of E. coli isolate 37 and K. pneumoniae isolate 24, which exhibited different genetic profiles, IS903 was present. In K. pneumoniae ST15 isolates, both ISEcp1 and IS903 were found flanking bla_CTX-M-15, facilitating its mobilization between plasmids and chromosomes. Integron analysis in E. coli isolates 49 and 37, and K. pneumoniae isolates 35, 40, 24, 7, 17, 29, and 43, revealed a class I integron in E. coli isolate 49, containing the dhfrV gene linked to trimethoprim resistance. To investigate plasmid dissemination mechanisms, plasmid incompatibility groups were identified using replicon typing. Five multiplex and three simplex PCR reactions were conducted targeting 18 replication origin groups. E. coli isolates 1, 2, 3, and 49 tested positive for IncFIA, IncFIB, and IncF, while E. coli isolate 37 harbours IncFIA and IncF. Among K. pneumoniae isolates, only isolate 7 carried IncN, with no positive results in others. These findings are consistent with previous research showing that IncFII plasmids are associated with the spread of blaCTX-M-15 in E. coli. At the same time, K. pneumoniae producers of CTX-M β-lactamase are commonly associated with IncFII, IncN, and IncL/M plasmid groups [9].

3.8 Plasmid DNA extraction

The plasmid DNA extraction technique developed by Kado and Liu [25] was used for E. coli isolates 49 and 37 and K. pneumoniae isolates 24, 35, 40, 7, 17, 29, and 43 to determine the presence of plasmids in bacterial isolates. It was possible to identify plasmids in all isolates studied, with more than one plasmid observed in E. coli isolates 49 and 37 and K. pneumoniae isolate 43 (Figure 5). However, since plasmid DNA molecules can exist in different conformations with varying degrees of supercoiling, these isolates may not contain several plasmids but rather several isoforms of the same plasmid, resulting in the visualization of several bands on the agarose gel. E. coli isolates 49 (carrying a KPC-2 carbapenemase) and 37 (carrying an extended-spectrum β-lactamase CTX-M-15) have two isoforms of the plasmid of the same size, with isolate 49 having a smaller form, approximately 9 kb, which is not found in isolate 37. In K. pneumoniae isolates 35 and 40, which are identical both by the previous typing methods and in terms of the resistance genes they carry (CTX-M-15 and SHV-28), it is possible to identify two plasmids of different sizes, with the size of the plasmid of isolate 40 being similar to that of isolate 17, which carries a CTX-M-15, SHV-1, and TEM-1. The plasmid of isolate 24, which carries the resistance genes CTX-M-15, SHV-12, and TEM-1, is superior to that of the other isolates. This investigation shows the diversity of plasmids found in the various isolates, plasmids that can acquire various resistance genes and serve as a vehicle for their spread [38]. It is possible to observe the chromosomal fragments and RNA.

image017.png

Figure 5. Visualization of plasmid DNA on 0.7% agarose gel

a: Visualization of plasmid DNA extracted from E. coli and K. pneumoniae; b: Visualization of plasmid DNA extracted from K. pneumoniae; M: GeneRuler^TM 1kb DNA ladder molecular mass marker.

To determine whether the identified resistance genes were plasmid-borne, plasmids were extracted, purified, and analyzed by PCR using DNA recovered from agarose gel. The extracted bands were designated as follows: 37.1 and 37.2 for E. coli 37 (37.1 being the highest molecular mass and 37.2 the lowest), 40.1 for K. pneumoniae 40, 24.1 for K. pneumoniae 24, 35.1 for K. pneumoniae 35, 7.1, 17.1, 29.1, and 43.2 (lowest molecular mass) for K. pneumoniae isolates 7, 17, 29, and 43, and 49.1, 49.2, and 49.3 for E. coli 49 (49.1 being the highest molecular mass and 49.3 the lowest). PCR was conducted to detect bla_CTX-M, bla_TEM, and bla_SHV genes, along with ISEcp1 and IS903 insertion sequences in plasmids 24.1, 37.1, 37.2, 40.1, 35.1, 7.1, 17.1, 29.1, and 43.2. Additionally, bla_KPC amplification was performed on plasmids 49.1, 49.2, and 49.3. The results showed that bla_TEM was successfully amplified in all tested samples, confirming its plasmid location. However, no amplification was detected for bla_CTX-M, bla_SHV, or the investigated insertion sequences, suggesting these genes may reside chromosomally rather than on plasmids. Such a transfer may have occurred because ISEcp1 can mobilize bla_CTX-M from plasmids to chromosomes. However, the absence of amplification does not definitively exclude plasmid localization, as potential DNA fragmentation during gel extraction may have affected primer binding. Conversely, bla_KPC was successfully amplified in plasmids 49.1, 49.2, and 49.3. These findings, particularly the detection of bla_TEM in 37.1 and 37.2 and bla_KPC in 49.1, 49.2, and 49.3, suggest the presence of different isoforms of the same plasmid rather than multiple distinct plasmids within these isolates. The PCR amplification results for selected samples are shown in Figure 6.

ping_mu_jie_tu_2025-07-31_140156.png

ping_mu_jie_tu_2025-07-31_140209.png

Figure 6. PCR products for the amplification of the 544 bp bla_CTX-M (a), 1058 bp bla_TEM (b), 886 bp bla_SHV (c), and bla_KPC (d) genes on 1% agarose gel

CC: positive control for the blaCTX-M gene; CT: positive control for the blaTEM gene; CS: positive control for the blaSHV gene; CK: positive control for the blaKPC gene. M: GeneRuler^TM 1kb DNA ladder molecular mass marker.