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The emergence of bla NDM particularly in Gram-negative bacteria is a burden on the health care system in developing countries. Hence, this study was initiated to screen New Delhi Metallo-β-lactamase (NDM)-producing Gram-negative bacterial strains from neonatal intensive care unit (NICU) of an Indian Hospital. A total of 18 bla NDM -producing isolates were detected in the present study. Out of 18 bla NDM variant isolates, 6 were Klebsiella pneumoniae , 4 Escherichia coli , 2 Enterobacter aerogenes , 1 Acinetobacter lwoffii , 1 Enterobacter cloacae , 3 Acinobacter baumannii, and 1 Cedecea davisae from NICU, showing resistance against all antibiotics, except colistin and polymixin. The transferability of resistance determinants was tested by conjugation. Transfer of bla NDM -producing strains was successful in all 18 strains. In the case of transconjugants, the minimum inhibitory concentration values were found to decrease. The bla NDM -producing isolates contained detectable plasmids of size 66, 38, and 6 kb. Plasmi/d-based replicon typing revealed the incompatibility types Inc (A/C, FIIA, FIC, K, F, W, FIA, P, X, FIB, B/O) in bla NDM -carrying isolates. This study revealed the outbreak of multiple variants of bla NDM (13 NDM-1, 4 NDM-5, and 1 NDM-7). Moreover, other resistance markers, viz. bla OXA-1 , bla C MY-1 , bla VIM-1 , and bla SHV-1 coassociated with bla NDM were also found. In this study, we reported NDM-producing C. davisae as a first report to the best of our knowledge. This study is an attempt to reveal the dissemination of bla NDM isolated from neonates in NICU and their efficient transferability among Gram-negative bacilli through horizontal gene transfer.

Objective: The aim of this study was to investigate the resistance mechanisms of carbapenem-resistant Enterobacteriaceae clinical strains recovered from Al Thawra University Hospital, Sana'a, Yemen. Methods: A total of 27 isolates showing decreased susceptibility to carbapenems were obtained from different clinical specimens in Al Thawra Hospital, Sana'a, Yemen. Strains were identified by Matrix Assisted Laser Desorption Ionization Time-Of-Flight spectroscopy. Susceptibility to antibiotics was determined by the disk diffusion method on Mueller Hinton agar. Carbapenemases-encoding genes, extended-spectrum β-lactamases (ESBLs), and plasmid-mediated quinolone resistance (PMQR) genes were screened by PCR. Bacterial isolates were typed by multilocus sequence typing (MLST). Results: Carbapenemase genes detection and sequencing showed that 18 (66.7%) isolates were Klebsiella pneumoniae (NDM-1, n  = 13; NDM-1 + OXA-48, n  = 3; OXA-48, n  = 1; OXA-232, n  = 1), 6 (22.2%) were Escherichia coli (NDM-5, n  = 3; OXA-181, n  = 2; OXA-48, n  = 1), and 3 (11.1%) were Enterobacter cloacae (NDM-1, n  = 1; OXA-181, n  = 2). In addition the ESBL gene bla CTX-M-15 was detected in 14 K. pneumoniae and 2 E. coli isolates, and the bla CTX-M-216 was found in 1 E. coli isolate. Fifteen isolates were PMQR positive including qnrB1 ( n  = 1), qnrS1 ( n  = 5), qnrS4 ( n  = 2), and aac-(6')-Ib-cr ( n  = 7). The MLST typing showed a diversity of sequence type (ST) clones including Escherichia coli ST410 (3), ST448 (2), and ST648; Enterobacter cloacae ST78 and ST270; and Klebsiella pneumoniae ST395 (2), ST309, ST23, ST35, ST1728, ST15, ST231, and ST1428. Conclusion: This study reports the first description of OXA-48-like-producing Enterobacteriaceae and NDM-5 enzymes in E. coli in Yemen.

The emergence of New Delhi metallo-β-lactamase 1 on carbapenemase-producing bacteria has raised a major worldwide public health concern. This study reports the dissemination of bla NDM-1 in carbapenem-resistant isolates that caused nosocomial infections in a tertiary hospital in Mexico City. Seven Enterobacter cloacae and three Klebsiella pneumoniae nosocomial isolates from the same time period harbored the bla NDM-1 gene. The resistance phenotype and the bla NDM-1 gene were transferred through conjugative plasmids belonging to the incompatibility group IncFIA of 85, 101, and 195 kb in E. cloacae and 95 and 101 kb in K. pneumoniae isolates. Restriction fragment length polymorphism analysis showed that bla NDM-1 was carried in similar plasmids with molecular sizes of 101 and 85 kb, each one in three isolates of E. cloacae and one of 101 kb on two isolates of K. pneumoniae . During a 9-month period, six of the seven isolates of E. cloacae analyzed harbored bla NDM-1 and belonged to clone E1. Similarly, over a 5-month period, two of the three K. pneumoniae isolates that harbored bla NDM-1 belonged to clone K1. These results demonstrate the horizontal transfer of bla NDM-1 between different bacterial species, dissemination of clones with high levels of resistance to carbapenems, and underscore the need for heightened measures to control their further spread.

Enterobacter cloacae complex is a common cause of hospital outbreaks. A retrospective and prospective molecular analysis of carbapenem-resistant clinical isolates in a tertiary care center demonstrated an outbreak of a German-imipenemase-1 (GIM-1) metallo-beta-lactamase-producing Enterobacter hormaechei ssp. steigerwaltii affecting 23 patients between 2009 and 2016. Thirty-three isolates were sequence type 89 by conventional multilocus sequence typing (MLST) and displayed a maximum difference of 49 out of 3,643 targets in the ad-hoc core-genome MLST (cgMLST) scheme (SeqSphere+ software; Ridom, Münster, Germany). The relatedness of all isolates was confirmed by further maximum-likelihood phylogeny. One clonal complex of highly related isolates (≤15 allele difference in cgMLST) contained 17 patients, but epidemiological data only suggested five transmission events. The bla GIM-1 -gene was embedded in a class-1-integron (In770) and the Tn21-subgroup transposon Tn6216 (KC511628) on a 25-kb plasmid. Environmental screening detected one colonized sink trap in a service room. The outbreak was self-limited as no further bla GIM-1 -positive E. hormaechei has been isolated since 2016. Routine molecular screening of carbapenem-nonsusceptible gram-negative isolates detected a long-term, low-frequency outbreak of a GIM-1-producing E. hormaechei ssp. steigerwaltii clone. This highlights the necessity of molecular surveillance.

A plasmid pCY-CTX carrying a phage-like backbone from an extensively drug-resistant Enterobacter cloacae strain Guangzhou-ECL001 (previously known as CY01) was identified in this study. By Illumina MiSeq 2 × 250-bp paired-end sequencing, de novo assembly, and PCR, full sequence of pCY-CTX was obtained. Plasmid pCY-CTX was a circular plasmid with a length of 116,700 bp, harboring 136 putative open reading frames with the average G + C content of 50.8%. The backbone of pCY-CTX showed high identity to previously reported phage-like plasmid pHCM2 and phage SSU5. In addition, pCY-CTX contained a distinctive ISEcp1-mediated Tn2 region with two resistance genes bla TEM-1 and bla CTX-M-3 . Transposition unit "ISEcp1- bla CTX-M-3 - orf477" was inserted into the Tn2 structure, dividing Tn2 into two parts. This represents the first identification of a plasmid carrying a phage-like backbone and a distinctive ISEcp1-mediated Tn2 region within bla TEM-1 and bla CTX-M-3 in clinical E. cloacae. The finding of phage-like regions located in plasmids provides a new perspective in gene transfer associated with antimicrobial resistance.

Extended-spectrum beta-lactamases (ESBL) and carbapenemase-producing organisms pose severe problems for hospitalized patients. In the absence of efficient sanitation and sewage disposal, the risks for transmission of hospital organisms into the community are high. Our objectives were to study the occurrence and mechanisms of resistance of multidrug-resistant gram-negative bacilli in two Lebanese hospital sewage treatment plants. Wastewater samples were collected, filtered, and cultivated on MacConckey+cefotaxime agars. ESBL, AmpC, metallo-β-lactamases (MBL), and Klebsiella pneumoniae Carbapenemases (KPC) production were phenotypically detected using plain Mueller Hinton agar plates, and plates impregnated with 5 mM EDTA, 10 mg/mL phenyl boronic acid, and 250 μg/mL cloxacillin (embedded). Temocillin discs were used for the presumptive detection of OXAs. ESBL, carbapenemase, outer membrane protein F (OMPF), and outer membrane protein C (OMPC) genes were detected using polymerase chain reaction. Pulsed-field gel electrophoresis (PFGE) was performed to study the clonality of Enterobacter cloacae isolates. In total, 32 and 38 Enterobacteriaceae were detected from Hospital 1 and Hospital 2, respectively. All Escherichia coli and Klebsiella spp. isolates were ESBL producers. AmpC reached 25% and 28.9% of all isolates. Only one Enterobacter cloacae isolate from one hospital showed full resistance to carbapenems. Molecular tests, however, detected NDM-1 in two strains of Enterobacter cloacae. PFGE results showed 80% similarity between these two strains. The isolation of NDM-1-producing Enterobacter cloacae from hospital wastewater occurred almost a year before the first case of carbapenem-resistant Enterobacter spp. was detected from a patient sample in the laboratory hospital. Understanding the local epidemiology of resistance in hospitals should include areas of potential resistance, such as wastewater and hospital environment.

A total of 72 consecutive and nonduplicate clinical extended-spectrum β-lactamase (ESBL)-producing Enterobacter cloacae isolates were collected from our hospital from 2012 to 2014 for analyzing the prevalence of plasmid-mediated quinolone resistance (PMQR) genes, 16S rRNA methyltransferase (16S-RMTase) encoding genes, and carbapenem-hydrolyzing β-lactamase (CHβL) genes, as well as integrons. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were carried out to characterize the genetic relatedness. The isolates mainly harbored bla CTX-M (n = 51, 70.8%) and bla SHV (n = 46, 63.9%) genes. High prevalence of PMQR genes qnrA1 (n = 24, 33.3%), qnrB (n = 4, 5.6%), qnrS1 (n = 2, 2.8%), and aac(6')-Ib-cr (n = 21, 29.2%) was observed. Furthermore, CHβLs IMP-4 (n = 6, 8.3%) and IMP-8 (n = 4, 5.6%), as well as class I integrons (n = 29, 40.3%), were found in the ESBL-producing E. cloacae isolates. PFGE revealed 69 pulsotypes. MLST distinguished 44 sequence types (STs) with ST124 (n = 7, 9.7%), ST50 (n = 3, 4.2%), ST45 (n = 3, 4.2%), and ST93 (n = 3, 4.2%) being the predominant STs. The results indicate a possible clonal transmission of ST124 isolates in the hospital that needs further surveillance. The genetic diversity of the other numerous distinctive STs indicates that most of the ESBL-producing E. cloacae in our hospital might arise through stepwise accumulations of multiple drug-resistance determinants in different clones.

Enterobacter cloacae is one of the most common carbapenem-resistant Enterobacteriaceae (CRE) global wide. Resistance to tigecycline, one of the few therapeutic options for CRE infections, in carbapenem-resistant E. cloacae is of clinical significance. Fourteen E. cloacae clinical isolates (EC1-EC14) co-resistant to tigecycline and carbapenems were studied. Two tigecycline-susceptible/carbapenem-resistant isolates (TS1-TS2) were used for comparison. Genotyping by pulsed-field gel electrophoresis and multilocus sequence typing identified seven pulsotypes and three sequence types (STs). All three STs belonged to the published international clones. Polymerase chain reaction (PCR) and sequence analysis revealed the coexistence of bla SHV-12 and bla IMP-8 in 11 EC isolates from five pulsotypes/two STs. Reverse transcription PCR demonstrated overexpression of the chromosomal AmpC-like β-lactamase in seven EC isolates (four pulsotypes/two STs) and TS1 (pulsotype F/ST78). Reduced expression of outer membrane protein C (OmpC) was found in three EC isolates (all pulsotype C/ST204), whereas reduced expression of OmpF was found in nine EC isolates (three pulsotypes/two STs) and TS2 (pulsotype G/ST114). Overexpression of the efflux pump AcrB was found in all EC isolates although three showed borderline significance. Multiple mechanisms jointly contributed to the observed co-resistance to tigecycline and carbapenems. Some international clones have infiltrated into Taiwan and acquired various resistance traits independently.

Extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-PE) have been described worldwide, but few reports focused on Burkina Faso. To assess the prevalence of digestive carriage of such bacteria in the community and in the hospital, 214 fecal samples, 101 from healthy volunteers and 113 from hospitalized patients without digestive pathology, were collected in Bobo Dioulasso, Burkina Faso economic capital, during July and August 2014. Stool samples were screened using ESBL agar plates. Strains were identified by mass spectrometry using the Biotyper MALDI-TOF. ESBL production was confirmed with the double-disc synergy test. Susceptibility was tested using the disk diffusion method on Müller-Hinton agar. The main ESBL genes were detected using multiplex PCR and bidirectional gene sequencing. Escherichia coli phylogenetic groups were identified using a PCR-based method. During the study period, prevalence of subjects with fecal ESBL-PE was 32% (69/214), 22% among healthy volunteers and 42% among inpatients. All but two ESBL, CTX-M-15 and ESBL-PE, were mostly E. coli (78%). Among the 60 ESBL-producing E. coli strains, 26% belonged to phylogenetic group D, 23.3% to group A, 20% to group B1, 6.6% to group B2, and 3.3% to the ST131 clone. Univariate analysis showed that history of hospitalization and previous antibiotic use were risk factors associated with ESBL-PE fecal carriage. In Burkina Faso, the prevalence of both healthy subjects from the community and hospitalized patients with fecal ESBL-PE is alarmingly high. This feature should be taken into consideration by both general practitioners and hospital doctors with regard to empirical treatments of infections, notably urinary tract infections.

The aim of this study was to investigate the infections due to OXA-48 carbapenemase-producing bacteria in tertiary hospitals in Kuwait (September 2011 to April 2013) and to determine the sequence types (STs) of the corresponding isolates. Eleven OXA-48 carbapenemase-producing Enterobacteriaceae isolates were recovered from patients treated in nine different hospitals in Kuwait. Susceptibility testing to eighteen antibiotics was done using the E-test. PCR assays were performed for the detection of genes encoding extended-spectrum β-lactamases (ESBLs) (blaCTX-M, blaSHV, and blaTEM) and carbapenemases (blaOXA-48, blaVIM, blaNDM, blaIMP, blaGIM, and blaKPC). STs were determined by Multilocus Sequence Typing. Seven Klebsiella pneumoniae, two Escherichia coli, one Enterobacter cloacae, and one Morganella morganii harbored the blaOXA-48 gene. The K. pneumoniae and E. coli belonged to seven and two different STs, respectively, which were not related to those reported from this region. The majority of the isolates carried either blaCTX-M or blaSHV genes and showed a multidrug-resistant phenotype, including resistance to tigecycline. Multidrug-resistant Enterobacteriaceae harboring the blaOXA-48 gene are emerging in Kuwait with different STs compared to those identified in other countries of the region. Detection of OXA-48-producing Enterobacteriaceae in Kuwait is important to prevent its rapid spread.

Emergence of carbapenem-resistant Enterobacteriaceae has become a substantial global health problem. The aim of this study was to analyze carbapenem-resistant isolates of Enterobacter cloacae that have emerged for the first time in the intensive care unit (ICU) at the University Hospital Centre Split, Croatia. The strains were selected in the period between June and August 2012, according to their susceptibility patterns to carbapenems. Resistant isolates were screened for metallo-β-lactamase (MBL) production with the use of the imipenem-EDTA disk synergy test, and positive findings were confirmed by PCR. The type of VIM β-lactamase gene was determined by sequencing of PCR products. The genetic relatedness was evaluated using pulsed-field gel electrophoresis analysis. The demographic and clinical data were retrospectively analyzed from medical records. Five patients were infected and one patient was colonized with a single clone of multidrug-resistant VIM-1-producing E. cloacae susceptible only to colistin. Three cases of lower respiratory tract infections, one case of bacteremia, and one case of intra-abdominal infection were identified. All cases were hospital-acquired after prolonged stay in ICU. All patients had serious underlying diseases and received a broad-spectrum antibiotic. Four patients died and two had unimprovable medical condition at the time of discharge from the hospital. MBL-producing E. cloacae can cause fatal infection in severely ill patients. Monoclonal outbreak highlights the need for continuous surveillance and good infection control practices to prevent further spread since the antibiotic therapy options for infections caused by such strains are strongly limited.

Extended-spectrum β-lactamase (ESBL) production is an important resistance mechanism among clinical isolates of Enterobacter cloacae. TEM, SHV, and CTX-M are the most common ESBL genotypes among Enterobacter spp. The main aims of this study were to determine the antimicrobial susceptibility pattern and to detect ESBL-encoding genes as well as intI1 genes. One hundred twenty isolates of E. cloacae were collected from hospitals of Tehran and Qazvin, Iran. The isolates were identified by standard laboratory methods and API 20E strips. ESBL screening was performed by the combined disk method. PCR and sequencing were conducted for detection of ESBL-encoding genes as well as intI1 genes. Clonal relatedness of ESBL-producing isolates was assessed by the enterobacterial repetitive intergenic consensus (ERIC)-PCR method. Of 120 isolates, 57 (47.5%) were characterized as multidrug resistant among those 48 (84.2%) isolates carried class 1 integron. Fifty-three (44.2%) isolates were found to be ESBL producers, in which bla(CTX-M-15) (60.4%) was the most common gene followed by bla(TEM-1) (32.1%), bla(TEM-169) (13.2%), and bla(SHV-12) (7.5%) either alone or in combination. Forty-four of the 53 (83.01%) ESBL-producing isolates were genetically unrelated. For the first time, this study describes the emergence of TEM-169, SHV-12, and CTX-M-15 ESBL genotypes in E. cloacae isolates in Iran.

Seventy-one isolates of Enterobacteriaceae (17 Escherichia coli, 50 Klebsiella pneumoniae, and 4 Enterobacter cloacae) producing extended-spectrum-β-lactamases (ESBLs) were collected between April 2008 and March 2010 in an intensive care unit and surgical ward of Tlemcen Hospital (West of Algeria). Sequencing identified the bla(CTX-M-15) determinant in 69 isolates and bla(CTX-M-3) in 2 isolates. None of the studied strains produced the class D carbapenemase OXA-48. Repetitive Extragenic palindromic polymerase chain reaction showed a high degree of genotypic diversity among E. coli strains and two major clonal populations of K. pneumoniae (CKp1 n=11 and CKp5 n=25) which were further identified as members of the multilocus sequence typing types (ST931) and (ST15), respectively. The ST15 isolates harbored more resistance genes and virulence factors than the ST931 isolates. The characterization of the spacer region between ISEcp1 and bla(CTX-M) for CTX-M-15 producers individualized two populations. One that derived from the CTX-M-3 under Algerian clinical context and one that is universally found. The dissemination of ESBLs in the studied Enterobacteriaceae isolates was mainly due to the epidemic clones of K. pneumoniae and to genetic transit of plasmids among unrelated strains.

In 2009, out of the 66 nonrepetitive Enterobacter cloacae collected at Charles Nicolle hospital in Tunisia, 44 were extended spectrum β-lactamase (ESBL) producers. The aim of the current study was to detect and characterize the genes encoding the ESBLs including blaTEM, blaSHV, and blaCTX-M groups by polymerase chain reaction and sequencing. Pulsed-field gel electrophoresis (PFGE) analysis was used to determine the genetic relatedness between isolates. All strains were susceptible to carbapenems. They were resistant to fluoroquinolones, gentamicin, tobramycin, and trimethoprim+sulfamethoxazole but variably resistant to netilmicin, amikacin, and tetracyclines. Sequence analysis of the polymerase chain reaction products revealed the presence of blaCTX-M-15 (39 strains), blaSHV-12 (6 strains), and blaSHV-27 (1 strain). The coexistence of two ESBLs was observed in two isolates harboring, respectively, SHV-12+CTX-M-15 and SHV-27+CTX-M-15. PFGE revealed 36 unrelated profiles. Diffusion of E. cloacae producing CTX-M-15 ESBL in our hospital is the consequence of dissemination of identical or related plasmids harboring the CTX-M-15 gene.

Four isolates of Klebsiella pneumoniae and one isolate of Enterobacter cloacae exhibiting resistance to most β-lactam antibiotics, including oxyimino-cephalosporins and carbapenems, were obtained from different patients among four hospitals in China. Pulsed-field gel electrophoresis demonstrated that all the K. pneumoniae isolates belonged to two clone patterns. Multilocus sequence typing showed that the four isolates of K. pneumoniae belonged to two sequence types: ST 23 and ST 351. Conjugation studies with Escherichia coli (EC600) resulted in the transfer of reduced carbapenem susceptibility compared with that of the original isolates. Plasmid restriction analysis and hybridization experiment showed that the five isolates of Enterobacteriaceae carried a common 50 kb bla(KPC-2)-encoding plasmid.

Tigecycline, a novel broad-spectrum glycylcycline antibiotic, is active against many gram-positive and gram-negative bacterial pathogens including most strains of Enterobacter cloacae. Recently, however, a few clinical strains of E. cloacae with decreased susceptibility to tigecycline were isolated. In this study, two tigecycline-susceptible mutants of E. cloacae, GC7696 and GC7697, were obtained by transposon mutagenesis of a tigecycline-resistant clinical isolate G946. Transposon insertions were mapped to either the acrA or acrB genes. Restoration of the original resistant phenotype occurred when GC7696 and GC7697 were transcomplemented with a plasmid harboring the intact acrAB region amplified from G946. Northern blot analysis of G946 and several other E. cloacae clinical strains that exhibited decreased susceptibility to tigecycline, revealed increased levels of the acrAB transcript. In addition, overexpression of acrAB correlated with increased expression of the ramA gene, whereas the expression of another transcriptional activator, marA, was not changed. These results suggest that decreased susceptibility to tigecycline in E. cloacae is the result of RamA-mediated overexpression of the AcrAB efflux pump.

The aim of this study was to search for plasmid-encoded quinolone resistance determinants QnrA and QnrS in fluoroquinolone-resistant and extended-spectrum beta-lactamase (ESBL)-producing enterobacterial isolates recovered in Sydney, Australia, in 2002. Twenty-three fluoroquinolone-resistant, of which 16 were also ESBL-positive, enterobacterial and nonrelated isolates were studied. PCR with primers specific for qnrA and qnrS genes and primers specific for a series of ESBL genes were used. A qnrA gene was identified in two ESBL-positive isolates, whereas no qnrS-positive strain was found. The QnrA1 determinant was identified in an Enterobacter cloacae isolate and in a carbapenem-resistant Klebsiella pneumoniae isolate, both of which expressed the same ESBL SHV- 12. Whereas no plasmid was identified in the E. cloacae isolate, K. pneumoniae K149 possessed two conjugative plasmids, one that harbored the qnrA and bla (SHV)-12 genes whereas the other expressed the carbapenemase gene bla (IMP-4). The qnrA gene, was located in both cases downstream of the orf513 recombinase gene and upstream of the qnrA1 gene, a structure identical to that found in sul1-type integron In36 and qnrA-positive strains from Shanghai, China. However, the gene cassettes of the sul1-type integrons were different. This study identified the first plasmid-mediated quinolone resistance determinant in Enterobacteriaceae in Australia.